Molecular principles of protein stability and protein-protein interactions

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Molecular principles of protein stability and protein-protein interactions Christofer Lendel School of Biotechnology Royal Institute of Technology

Stockholm 2005

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© Christofer Lendel Stockholm 2005 Royal Institute of Technology School of Biotechnology AlbaNova University Center SE-106 91 Stockholm Sweden Printed by Universitetsservice US-AB Drottning Kristinas väg 53B SE-100 44 Stockholm Sweden ISBN 91-7178-189-7 Cover illustration: Ribbon representation of the Z:ZSPA-1 complex structure.

iii Christofer Lendel (2005). Molecular principles of protein stability and protein-protein interactions. School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden

Abstract Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z domain variants with 13 randomised positions at the immunoglobulin Fc-binding surface. This thesis aims to describe the principles for molecular recognition in two protein-protein complexes involving affibody proteins. The first complex is formed by the ZSPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 µM). The second complex consists of two affibodies: ZTaq, originally selected to bind Taq DNA polymerase, and anti-ZTaq, an anti-idiotypic binder to ZTaq with a Kd ~0.1 µM. The basis for the study is the determination of the three-dimensional structures using NMR spectroscopy supported by biophysical characterization of the uncomplexed proteins and investigation of binding thermodynamics using isothermal titration calorimetry. The free ZSPA-1 affibody is a molten globule-like protein with reduced stability compared to the original scaffold. However, upon target binding it folds into a well-defined structure with an interface topology resembling that displayed by the immunoglobulin Fc fragment when bound to the Z domain. At the same time, structural rearrangements occur in the Z domain in a similar way as in the Fc-binding process. The complex interface buries 1632 Å2 total surface area and 10 out of 13 varied residues in ZSPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of ZSPA-1 revealed a native-like overall structure with increased dynamics in the randomised regions (helices 1 and 2). These features were reduced when replacing some of the mutated residues with the corresponding wild-type Z domain residues. The nature of the free ZSPA-1 affects the thermodynamics of the complex formation. The contribution from the unfolding equilibrium of the molten globule was successfully separated from the binding thermodynamics. Further decomposition of the binding entropy suggests that the conformational entropy penalty associated with stabilizing the molten globule state of ZSPA-1 upon binding seriously reduces the binding affinity. The ZTaq:anti-ZTaq complex buries in total 1672 Å2 surface area and all varied positions in anti-ZTaq are directly involved in binding. The main differences between the Z:ZSPA-1 and the ZTaq:anti-ZTaq complexes are the relative subunit orientation and certain specific interactions. However, there are also similarities, such as the hydrophobic interface character and the role of certain key residues, which are also found in the SPA:Fc interaction. Structural rearrangements upon binding are also common features of these complexes. Even though neither ZTaq nor anti-ZTaq shows the molten globule behaviour seen for ZSPA-1, there are indications of dynamic events that might affect the binding affinity. This study provides not only a molecular basis for affibody-target recognition, but also contributions to the understanding of the mechanisms regulating protein stability and proteinprotein interactions in general. Keywords: affibody, binding thermodynamics, induced fit, molten globule, NMR spectroscopy, protein engineering, protein-protein interactions, protein stability, protein structure. © Christofer Lendel, 2005

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Main References This thesis is based on the following papers, referred to in the text by their Roman numerals. I.

Lendel, C.*, Wahlberg, E.*, Berglund, H., Eklund, M., Nygren, P.-Å. and Härd, T. (2002). 1H, 13C and 15N resonance assignments of an affibody-target complex. J. Biomol. NMR 24: 271-272.

II.

Wahlberg, E.*, Lendel, C.*, Helgstrand, M., Allard, P., Dincbas-Renqvist, V., Hedqvist, A., Berglund, H., Nygren, P.-Å. and Härd, T. (2003). An affibody in complex with a target protein: Structure and coupled folding. Proc. Natl. Acad. Sci. USA 100: 3185-3190.

III.

Lendel, C., Dincbas-Renqvist, V., Flores, A., Wahlberg, E., Dogan, J., Nygren, P.-Å. and Härd, T. (2004). Biophysical characterization of ZSPA-1 - A phagedisplay selected binder to protein A. Protein Sci.. 13: 2078-2088.

IV.

Dincbas-Renqvist, V., Lendel, C., Dogan, J., Wahlberg, E. and Härd, T. (2004). Thermodynamics of folding, stabilization, and binding in an engineered proteinprotein complex. J. Am. Chem. Soc. 126: 11220-11230.

V.

Dogan, J.*, Lendel, C*. and Härd, T. (2005). NMR assignments of the free and bound-state protein components of an anti-idiotypic affibody complex. Submitted

VI.

Lendel, C.*, Dogan, J.* and Härd, T. (2005). Structural basis for molecular recognition in an affibody:affibody complex. Manuscript.

* Joint first authors contributing equally to the work.

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Contents Introduction ................................................................................................................................................ 1 1

Proteins ............................................................................................................................................... 1

2

Protein structure................................................................................................................................ 2

3

Forces in protein structures ............................................................................................................ 5

4

5

6

7

3.1

Electrostatic interactions ........................................................................................................ 7

3.2

van der Waals interactions...................................................................................................... 7

3.3

Hydrogen bonds ...................................................................................................................... 8

3.4

Hydrophobic interactions.......................................................................................................9

3.5

Structure-based calculations of thermodynamic properties ...........................................10

Protein stability................................................................................................................................11 4.1

Forces in the folded state .....................................................................................................11

4.2

Formation of secondary structure ......................................................................................12

4.3

Forces in the unfolded state.................................................................................................13

4.4

Unfolding of proteins............................................................................................................13

4.5

Molten globules......................................................................................................................14

Protein-protein interactions ..........................................................................................................16 5.1

Classification...........................................................................................................................16

5.2

Structural aspects of the interface.......................................................................................17

5.3

Driving-forces of complex formation................................................................................17

5.4

The role of individual residues ............................................................................................18

5.5

Affinity and specificity ..........................................................................................................19

5.6

Conformational change ........................................................................................................20

Protein engineering.........................................................................................................................21 6.1

Rational design .......................................................................................................................21

6.2

Combinatorial methods ........................................................................................................23

6.3

Scaffolds for engineered binding proteins.........................................................................23

6.4

Staphylococcal protein A......................................................................................................24

6.5

Affibodies................................................................................................................................28

Experimental methods to study protein structure ....................................................................30 7.1

Size exclusion chromatography ...........................................................................................30

7.2

Isothermal titration calorimetry...........................................................................................30

7.3

UV-VIS spectroscopy ...........................................................................................................31

ix 7.4

Circular dichroism spectroscopy.........................................................................................31

7.5

Nuclear magnetic resonance spectroscopy........................................................................32

7.6

Other methods to determine 3D structures......................................................................35

Present investigation ................................................................................................................................37 8

Structural investigation of the Z:ZSPA-1 complex (I, II)............................................................38

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Biophysical characterization of the ZSPA-1 affibody (II, III, IV) .............................................43

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Investigation of the thermodynamics of the Z:ZSPA-1 complex formation (IV) .............50

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Investigation of the ZTaq:anti-ZTaq complex (V, VI) ............................................................53

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Concluding remarks ...................................................................................................................61 12.1

Affibody-target recognition .................................................................................................61

12.2

Protein stability.......................................................................................................................62

12.3

Protein-protein interactions .................................................................................................62

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Acknowledgements ....................................................................................................................63

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References....................................................................................................................................64

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Molecular principles of protein stability and protein-protein interactions

Abbreviations 2D

Two-dimensional

3D

Three-dimensional

ANS

8-anilino-1-naphtalenesulfonic acid

Anti-ZTaq

ZTaq – binding affibody anti-ZTaq S1-1 (clone 16:4).

CD

Circular dichroism

CPMG

Carr-Purcell-Meiboom-Gill

Fab

Fragment, antigen binding

Fc

Fragment, crystallisable

GuHCl

Guanidine hydrochloride

HSQC

Heteronuclear single quantum correlation spectroscopy

Ig

Immunoglobulin(s)

ITC

Isothermal titration calorimetry

Kd

Dissociation constant

NMR

Nuclear magnetic resonance

NOE

Nuclear Overhauser effect

NOESY

Nuclear Overhauser enhancement spectroscopy

R1

Longitudinal (spin-lattice) relaxation rate

R2

Transverse (spin-spin) relaxation rate

Rex

R2 contributions from chemical exchange

SEC

Size exclusion chromatography

SPA

Staphylococcal protein A

Taq

Thermus aquaticus

TMAO

trimethylamine-N-oxide

TOCSY

Total correlation spectroscopy

ZSPA-1

SPA-binding affibody

ZTaq

Taq DNA polymerase-binding affibody ZTaq S1-1.

Christofer Lendel

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Introduction 1 Proteins Proteins are of outstanding importance and diversity among all biological macromolecules. Their functions range from structural scaffolds (such as collagen) to highly specific enzymes that regulate and catalyse chemical reactions, including functional aspects such as transport and storage (e.g. hemoglobin), signalling (e.g. transmission of nerve impulses) and cellular defence (antibodies). Their medical importance is reflected by the fact that the majority of all drug targets are proteins. The key to understanding cellular function and malfunction is thus to understand proteins on molecular level. The importance of proteins was recognized already by Berzelius, who dictated the name ‘protein’, in 1838 (Stryer 1995). The name originates from the Greek word protos, meaning ‘prime’ or ‘first’. He also suggested that enzymes are cellular catalysts. In 1902 Fischer and Hofmeister independently concluded that proteins are composed of a covalent chain of amino acids (Dill 1990; Tanford and Reynolds 2001). The X-ray crystallography studies of amino acids, peptides and fibrous proteins conducted by Pauling and Corey during the 1930s contributed substantially to the understanding of protein structure and led to the proposal of the secondary structure elements in 1951 (Pauling and Corey 1951a-c; Pauling et al. 1951). In 1953 Sanger presented the amino acid sequence of insulin (Sanger and Thompson 1953a,b). Five years later, a major breakthrough came when the first three dimensional structure of a globular protein, myoglobin, was determined (Kendrew et al. 1958). With the molecular basis available, the development of biological science and the understanding of cellular processes accelerated considerably. Along with these findings the understanding of how proteins are coded on the DNA level has been of great importance. These mechanisms were elucidated mainly during the 1960s with the structural insights of Watson and Crick as starting point (Stryer 1995). In the 1970s the recombinant DNA technique rapidly developed with the main achievements being recombinant expression of proteins in foreign hosts, rapid determination of protein sequences on the DNA level and the possibility to modify and engineer proteins using more or less rational approaches.

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Despite the fact that proteins have been investigated for more than 150 years and the rapid development of the field in recent years, the molecular mechanisms that regulates how proteins adopt their three dimensional structures and how they recognize and bind to other biomolecules are not completely understood.

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Protein structure

Protein molecules are linear polymers of amino acids, which are further organized in up to four structural levels (figure 1). The investigation presented in this thesis concern all four levels in one way or another and I will therefore give a brief description of their structural properties.

L S Q Q R E E

N

I N

A

F

L N

a

b

c

d

Figure 1. Illustrations of (a) primary-, (b) secondary-, (c) tertiary-, and (d) quarternary protein structure.

The importance of the amino acid sequence, also denoted the primary structure, is not possible to overestimate since all properties of the protein are encoded in the sequence, including fundamental aspects as overall structure, stability and specificity of interactions with other biomolecules (Anfinsen 1973). The 20 amino acid building blocks differ only in the nature of the side chains, which basically can be classified as non-polar, polar or charged. The amino acids are connected via peptide bonds (figure 2) and it was early shown by Pauling and coworkers that these bonds are planar and that they have double bond character (Pauling et al. 1951). Another notable property of the peptide backbone is the L configuration of the chiral Cα atom in all naturally occurring amino acids, which means that there is a basic stereochemical asymmetry in proteins.

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As mentioned in chapter 1, Pauling and colleagues were also first to describe the elements of secondary structure (Pauling and Corey 1951a-c; Pauling et al. 1951). At least three types of helical structures can be found in proteins. The α-helix is formed by backbone amide-tocarbonyl hydrogen bonding between each n, n+4 amino acid residue pair resulting in a helical structure with 3.6 amino acid residues per turn (Branden and Tooze 1999). A tighter variant, denoted 310 helix, with hydrogen bonding from n to n+3 can sometimes be observed. There is also an n, n+5 variant called the π-helix. These helical structures are normally right-handed due to the L configuration of the amino acids. The first and last residues in a helix have no backbone partners for hydrogen bonding; instead side chain groups are recruited in these positions in so-called N- and C-caps. The aligned hydrogen bonds cause an overall dipole moment of the helix with a positive partial charge at the N-terminus and a negative partial charge at the C-terminus. The partial charges can be as large as 0.5-0.7 unit charges (Branden and Tooze 1999).

a

R2

H

b H

ψ R1

χ1

φ

N R2 H

N

R1

O

H

O Residue

Residue

Residue

Residue

Figure 2. A dipeptide with a connecting peptide bond represented as (a) ball and stick model and (b) chemical drawing. R1 and R2 represent side chains. The φ, ψ and χ1 dihedral angles are shown in (a).

Several extended peptide strands can interact with each other creating pleated sheet structures. These so-called β-sheets can be parallel, anti-parallel or mixed, referring to the relative direction of the peptide chains in the strands. The amide groups of the backbone are hydrogen bonded to the carbonyls in the adjacent strand. This arrangement allows all potential hydrogen bond donors and acceptors to be satisfied, except for those at the edges.

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Secondary structure elements are connected by loops that are more or less ordered. Short loops, so-called turns, often have well-defined geometry while longer loops are flexible. Long disordered loops and flexible terminal parts are often referred to as random coil structure. Due to steric clashes, only certain combinations of the φ and ψ dihedral angles (figure 2) are allowed. These values were calculated by Ramachandran and are often presented as a Ramachandran diagram (Ramachandran and Sasisekharan 1968). Different types of secondary structure correspond to well-defined regions in this diagram (figure 3). In general, glycines can adopt a wider range of conformations while prolines, on the other hand, increase the local rigidity of the peptide backbone. In a similar way there are restrictions for the χi angles of the side chains (figure 2). 180

β−sheet

ψ (°)

90

0 left-handed helix

helix -90

-180 -180

-90

0

φ (°)

90

180

Figure 3. Ramachandran diagram adopted from PROCHECK-NMR (Laskowski et al. 1996). The black regions are the most favourable for each type of secondary structure. Additional allowed regions are grey.

The elements of secondary structure pack together into more complex tertiary structures. The basic geometric aspects of this process have been described by Chothia and co-workers (Chothia et al. 1977; Chothia et al. 1981). In addition, the actual nature of the side chains will affect the structure. Central for the formation of tertiary structures is the packing of hydrophobic side chains to obtain a compact core with high structural and chemical

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complementarity. The forces governing this process will be further described in chapter 4. The tertiary structure is often divided into more or less independent units, called domains. Many proteins are fully functional as monomeric molecules while others associate with additional polypeptide chains of the same or different composition. Such assemblies of quarternary structure can be strictly structural or associated with the actual function. The mechanisms of these intermolecular interactions will be further described in chapter 5.

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Forces in protein structures

The aim to describe proteins on a molecular level requires understanding of the fundamental forces involved and how they interact to regulate biomolecular processes. I will in this chapter describe the foundations upon which the following discussion in the thesis is based. The direction of a chemical reaction is at constant pressure and temperature determined by the relative difference in Gibbs free energy (∆G°) between the end states. The free energy is related to the equilibrium constant (K) of the reaction: ∆G° = -RT ln (K)

(1)

where R is the gas constant and T the temperature in Kelvin. The free energy has two components, the enthalpy (∆H°), which in chemical systems is related to bonds and interactions, and the entropy (∆S°), which is associated with the degrees of freedom. ∆G° = ∆H° - T∆S°

(2)

Another important thermodynamic property is the heat capacity (∆Cp°), which is defined as the temperature derivative of the enthalpy or, in a more physical sense, a substance's energy storage capacity. In a region where the heat capacity is not temperature dependent the free energy can be expressed as

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Molecular principles of protein stability and protein-protein interactions ∆G° = ∆H°ref – T∆S°ref + ∆Cp° (T – Tref – T ln(T / Tref))

(3)

where ∆H°ref and ∆S°ref are the changes in enthalpy and entropy at the reference temperature Tref or ∆G° = ∆Cp° (T – TH – T ln(T/TS))

(4)

in which TH and TS are defined by ∆H°(TH) = 0 and ∆S°(TS) = 0, respectively. The values of these temperatures are, by themselves, characteristics for the mechanisms of the studied process. In general, the energetics of protein reactions is the sum of many small contributions giving large intrinsic free energies of both the starting state and the final state. The resulting free energy differences are often small and sensitive to environmental conditions. This makes simulations and computational analysis complex and inaccurate. The situation is further complicated by the surrounding water. As will be described, water and other solvent molecules are, in fact, responsible for many of the characteristics of proteins. Most of the forces determining protein structure at levels above the primary structure are of non-covalent nature (figure 4). The disulfide bridges formed by adjacent cysteine residues are one exception. There are also a number of covalent modifications that occur in vivo, such as phosphorylation. Even though these modifications have important regulatory functions they are not fundamental for protein structure in general. The strength of attractive non-covalent forces depends of the nature of the interacting partners, the distance between them and the properties of the surrounding. Charge-charge interactions have, for instance, energies proportional to 1/(D·r) (Atkins 1994), where D is the dielectric constant of the medium and r is the distance between the ions, while the forces between two randomly oriented permanent dipoles are proportional to 1/(D·r6), which means that they are less important at long distances. These simple relations indicate that it, given the three-dimensional structure of protein, would be straightforward to compute the total interaction energy, but in reality an accurate value of D is not easily obtained. D for water is 80 while it inside a protein is 2-4 (Fersht 1999). This means that D can vary with a factor 40 between different sites in a protein.

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b

+

e d a

c

f Figure 4. Examples of forces in and between protein molecules. (a) Backbone amide to carbonyl hydrogen bond. (b) Hydrogen bond between a protein side chain and water molecules. (c) Salt-bridge (charge-charge interaction) between protein side chains. (d) van der Waals interactions between nonpolar parts. (e) Ordering of water molecules around non-polar surface side chains. (f) Covalent disulfide linkage.

3.1 Electrostatic interactions Electrostatic interactions between pairs of charges can have intrinsic energies of 50 kcal mol-1 (Atkins 1994). However, due to the surrounding water, the net effect for ion pair interactions in proteins is typically a few kcal mol-1 (Dill 1990). Since the transfer of an ion pair into a low dielectric environment is highly unfavourable, charge-charge interactions are usually restricted to the protein surface (Honig and Nicholls 1995). However, once buried, the formation of a salt-bridge will be favourable and contribute an important source for specificity in biomolecular processes.

3.2 van der Waals interactions The attractive part of van der Waals interactions originates from momentaneous mutual induced dipoles and shows the same r-6 distance dependence as randomly oriented dipoles (Atkins 1994; Fersht 1999). The strengths of these interactions are also highly dependent on the polarizability of the atoms. It can for instance be noted that the polarizability of a methylene group is almost twice that of a hydroxyl group, indicating that van der Waals

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contacts between aliphatic parts will be more important than between hydroxyl groups or between protein and water (Fersht and Dingwall 1979; Fersht 1999). There is also a repulsive part of the van der Waals interaction that becomes important at short distance (r-12 distance dependence). The source of this force is the electron clouds of the atoms coming too close. The sum of these contributions give a well shaped potential where the distance giving the energy minimum is the van der Waals radius. The well is quite flat which means that small deviations from the ideal radius will not result in large energy penalties. Even though each van der Waals interaction is weak, typically 0.1 kcal mol-1, they are many and cumulative, resulting in a significant energy contribution.

3.3 Hydrogen bonds Hydrogen bonds can be formed between a donor, consisting of a partially charged hydrogen atom bound to an electronegative atom, and an electron donating acceptor. The distance between the hydrogen atom and the acceptor will be slightly shorter than the corresponding van der Waals contact due to the electrostatic nature of the bond (Fersht 1999). The intrinsic strength of a hydrogen bond is typically 5-10 kcal mol-1 (Fersht 1987; Atkins 1994). In proteins, however, the strength must always be evaluated relative to possible hydrogen bonds to water. In general, the enthalpic contributions for hydrogen bonding to water or another part of the protein are of the same magnitude, but the favourable entropy contribution of releasing bound water molecules often makes the inter-protein hydrogen bonds slightly more favourable. If there is no competition with water, like in the core of a protein, a hydrogen bond can use its full strength. However, as for the salt-bridges, the highly unfavourable energy of burying polar residues must be considered for the net effect of such a hydrogen bond. Furthermore, the formation of on average 3.4 hydrogen bonds per water molecule in its liquid state is the origin of many of the peculiar properties of water (Stryer 1995). In the classical definition of hydrogen bonds, the electronegative atoms of the donor and the acceptor are oxygen, nitrogen or fluorine. Recently, more exotic donors and acceptors have been recognized. For instance, carbon bonded hydrogen atoms (especially CαH) as donors and aromatic π-systems as acceptors (Derewenda et al. 1995; Weiss et al. 2001; Shi et al. 2002). The strengths of such hydrogen bonds are assumed to be weaker than for the classical bonds (Vargas et al. 2000; Scheiner et al. 2001), but frequent occurrence could still contribute to the

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net energy difference. These findings indicate that hydrogen bond and van der Waals interactions represent a continuum rather than two separate interaction classes.

3.4 Hydrophobic interactions The importance of hydrophobic interactions in biological systems was realized in the 1920s and 1930s from studies of the formation of lipid micelles and bilayers and how proteins associated with these (Southall et al. 2002). In 1954 Kauzmann introduced the term ‘hydrophobic bond’ and argued for its role for protein stability (Kauzmann 1954; Kauzmann 1959). Hydrophobic interactions originate from two contributions (Fersht 1999): i) the relative strength of the van der Waals interactions between hydrophobic parts compared to the corresponding interactions to water and polar species and ii) the propensity to hide hydrophobic surfaces from water contact due to the hydrophobic effect. The hydrophobic effect can easily be illustrated as the mechanism that makes oil and water separate, but the true molecular mechanism still remains a mystery. The origin of the effect is the peculiar thermodynamics of mixing oil and water noted by Edsall and Butler in the 1930s (Edsall 1935; Butler 1937). The thermodynamics is characterized by unfavourable excess entropy (the entropy left after subtracting the mixing entropy) at room temperature and a large increase in heat capacity for moving a non-polar solute into water (Dill 1990). Baldwin further noted from experimental data of small organic molecules that there seem to be specific values of TH ≈ 25 °C and TS ≈ 113 °C associated with the hydrophobic effect (Baldwin 1986). Interestingly, many biological processes show a constant ∆Sº/∆Cpº ratio that extrapolates to a value of TS ≈ 114 °C (Sturtevant 1977). The unfavourable entropy is a sign of increased order in the system and the change in heat capacity indicates a phase transition, in analogy with water-to-ice. These observations led Frank and Evans to propose the ‘iceberg’ model in 1945 (Frank and Evans 1945). They suggested that the water molecules surrounding a non-polar solute sacrifice motional freedom to keep the number and quality of the hydrogen bonds. At higher temperatures the increased mobility is exchanged for weaker hydrogen bonding. In analogy with the melting of ice, this transition provides an energy storage mechanism and is the reason for the large change in heat capacity. Neither this model nor other theories, for instance based on the energetic cost to create cavities in small molecule solvents, have so far provided quantitative explanations of the hydrophobic effect (Southall et al. 2002) and the understanding of the molecular basis is further complicated by the possibility of enthalpy-

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entropy compensation to minimize the free energy in water (Dunitz 1995; Grunwald and Steel 1995). In order to develop empirical relations, the transfer energies of amino acids and other small molecules from organic solvents, gas phase or crystalline state to water have been studied (Dill 1990; Fersht 1999; Southall et al. 2002). Even though the results are highly dependent on the choice of protein core-like phase, these experiments indicate that the hydrophobic effect contributes 20-30 cal mol-1 per Å2 buried surface (Hermann 1972; Chothia 1974; Reynolds et al. 1974).

3.5 Structure-based calculations of thermodynamic properties In the late 1980s Record and colleagues showed that transfer of hydrocarbons to water and protein unfolding have similar correlations between ∆Cpº and exposure of hydrophobic surface (Spolar et al. 1989; Livingstone et al. 1991). The model was soon expanded to include also polar contributions, and empirical relationships for obtaining thermodynamic parameters for protein folding from structure were developed (Murphy and Freire 1992; Spolar et al. 1992). These relationships have also shown to be valid for other processes such as protein-protein (Gomez and Freire 1995) and protein-DNA interactions (Spolar and Record 1994). For a detailed description of the methods see for instance (IV; Murphy 1999; Edgcomb and Murphy 2000; Lavigne et al. 2000). The requirements for accurate reproduction of the experimental thermodynamics are high quality structures of all states and that no extraordinary mechanisms, like binding of a metal ion or other coupled equilibria, are involved (IV; Bergqvist et al. 2004). However, even if all those criteria are fulfilled, there are cases where calculated data do not agree with experimental (Frisch et al. 1997; Henriques et al. 2000).

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Protein stability

Protein stability is determined by the driving force (i.e. the free energy difference) for proteins to populate the folded state relative more unfolded states. It is a topic closely related to protein folding, but while the main question in protein stability is ‘why’, the folding research also concerns ‘how’ and ‘how fast’. Since the latter questions are not really in the scope of my work, I will only discuss them briefly. Further introduction to protein folding can be found in e.g. (Daggett and Fersht 2003; Onuchic and Wolynes 2004; Lindorff-Larsen et al. 2005). The mechanisms by which proteins fold into three-dimensional structures are still not fully understood. Since the process, provided the right conditions, is spontaneous, the structure must be coded in the primary structure (Anfinsen 1973). The code is, however, quite robust, allowing for many mutations, as long as certain key residues are left intact. The stabilizing energy difference in proteins results from a very delicate balance (5-30 kcal mol-1) involving contributions from all the types of interactions described in the previous section (Dill 1990).

4.1 Forces in the folded state In 1936 Mirsky and Pauling presented a theory in which protein folding was mainly driven by hydrogen bonds and salt-bridges (Mirsky and Pauling 1936). The role of salt-linkages was discarded by Jacobsen and Linderstrøm-Lang (Jacobsen and Linderstrøm-Lang 1949) and when Pauling and colleagues in 1951 published their secondary structure suggestions (Pauling and Corey 1951a-c; Pauling et al. 1951), the results were based on the assumption of hydrogen bonds as the dominating force. This view was questioned by Kauzmann in 1954 (Kauzmann 1954; Kauzmann 1959), arguing that the energy difference between protein-protein and protein-water hydrogen bonds was too small. Instead he proposed the hydrophobic effect as major driving force, a suggestion that was further strengthened from structural data of myoglobin a few years later (Kendrew et al. 1958). The determination of protein structures has also once and for all discarded the electrostatic interactions (ion-pairs) as a dominant force for stability. Salt-bridges in proteins are not common enough to provide the required energy for the entire stabilization, which is not the same thing as they being unimportant. Several investigations have, in fact, demonstrated the influence of electrostatic forces for protein stability (Grimsley et al. 1999; Spector et al. 2000; Sanchez-Ruiz and Makhatadze 2001; Perl and Schmid 2001).

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Beside the high resolution structures there are several strong indications that the hydrophobic effect is the major driving force for globular collapse and formation of tertiary structure. For instance, the large ∆Cp° for unfolding, the notion that non-polar solvents denature proteins, the fact that salt affects protein stability according to the Hofmeister series and the high degree of structural conservation seen for the hydrophobic cores in proteins (Dill 1990).

4.2 Formation of secondary structure Considering the atomic arrangements in the secondary structure elements one could suspect that hydrogen bonds would be the most important force. However, the dominating contribution is in fact the hydrophobic effect and hydrogen bonding rather represents a way to compensate for burial of polar groups (Branden and Tooze 1999). The intrinsic propensities for the different amino acids to form elements of secondary structure have been investigated (Dill 1990; Fersht 1999). There is, however, an important difference between these measurements and the actual situation in a protein. The packing of secondary structure elements against other parts of the protein changes the propensities and introduces a positiondependence for the residues (Xiong et al. 1995). Beside the change in accessibility of hydrophobic residues, the stability of helices is affected by conformational restrictions of the side chains relative the unfolded state, the electrostatic compensation of the helix dipole moment (capping) and the hydrogen bonds (Horovitz et al. 1992). In addition, specific side chain interactions can further increase the stability. Experimental data indicate that alanine is the most suitable residue in central positions in helices (Lyu et al. 1990; O'Neil and DeGrado 1990; Horovitz et al. 1992). At the edges, residues that provide hydrogen bonding possibilities (including glycine that allows hydrogen bonding of water) but do not cause repulsion with the helix dipole are preferred (Serrano et al. 1992). β-sheet structures are closely associated with formation of tertiary structure and the major driving force is thus the hydrophobic effect. However, there are indications of intrinsic propensities for β-strand formation for residues like tyrosine, isoleucine and phenylalanine while glycine and proline seem to be strongly disfavoured (Kim and Berg 1993; Minor and Kim 1994a; Smith et al. 1994). The chemical basis for this preference is not obvious and the

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stability is as well dependent of the nature of the adjacent side chains (Minor and Kim 1994b; Smith and Regan 1995). Moreover, there is evidence that certain residues are preferred in reverse turns and that this propensity is related to local interactions rather than the overall fold. For instance, the stability of a β-turn formed by the peptide YPXDV was investigated for different amino acid residues in position X. The results were found to correlate well with the amino acid composition in turns derived from crystal structures (Dyson et al. 1988). The highest stability was observed with a glycine residue in position X, followed by Tyr, Asn, Asp and Phe.

4.3 Forces in the unfolded state The unfolded state is favoured by high conformational entropy, which was recognized already by Mirsky and Pauling in 1936 (Mirsky and Pauling 1936). The dramatically reduced entropy of a folded protein is enthalpically compensated by different kinds of interactions. For the surrounding water, the situation is the reversed. The unfolded protein induces higher order in the water structure due to the hydrophobic effect, but it also provides additional hydrogen bonding possibilities to the protein backbone. Furthermore, the unfolded state is not completely random coil and intra-protein interactions are transiently made and broken. The stabilizing effects of disulfide bridges can be understood in terms of reduced conformational entropy of the unfolded state due to the covalent linkage. This approach is frequently used by Nature to stabilize small peptides, for instance in snake venom (Kobayashi et al. 1991; Trabi and Craik 2002). Another important role of disulfide bonds is to connect the protein subunits, for instance in antibodies (Branden and Tooze 1999).

4.4 Unfolding of proteins Unfolding of proteins is in general reversible but the unfolded state has an increased propensity for aggregation, precipitation and degradation, which can cause non-reversibility. Due to the delicate energy balance in protein stability, the populations of the folded and unfolded states can easily be manipulated. Common ways of doing so are changing temperature, pH or pressure or adding different co-solvents (Fersht 1999). Unfolding by heat is mainly due to the conformational entropy becoming more important at high temperatures,

14


as can be understood from Eq. 2. Since certain groups in the protein can have different pKa values in the folded and the unfolded states, changes in pH can alter the relative stability of these states. At extreme pH values, high concentrations of equal charges can destabilize the folded state due to repulsive forces. Increased external pressure will favour the more compressible state, which is the unfolded state. Considering the importance of the solvent properties for protein reactions in general and protein stability in particular, it is obvious that addition of co-solvents will affect the folding equilibrium. Some substances, known as denaturants, decrease the stability while others, for instance so-called osmolytes, increase the stability. The molecular mechanisms for these effects are either direct contacts between the protein and the co-solvent or indirect effects on the structural properties of water. Denaturants like guanidine hydrochloride (GuHCl) and urea (figure 5a,b) have been shown to bind to the unfolded state, indicating a direct contact mechanism (Schellman 1994; Zou et al. 1998). Osmolytes, on the other hand, seem to work through preferential exclusion of water due to a solvophobic effect of the protein backbone (Gekko and Timasheff 1981; Lee and Timasheff 1981; Bolen and Baskakov 2001). The molecular origin of this effect is however not completely understood. Molecular dynamics simulations from Zou and co-workers indicated a mechanism where the osmolyte trimethylamine-N-oxide (TMAO, figure 5c) increases the order of the surrounding water molecules (Zou et al. 2002). From transfer experiments of cyclic dipeptides they further noted an enthalpic nature of the stabilizing effect, suggesting the preferential exclusion of water molecules is due the energetic cost of breaking of water-water hydrogen bonds.

4.5 Molten globules Many proteins fold without populating any stable intermediate states in so-called two-state reactions. Other folding pathways include more or less structured intermediates. An important class of intermediates is the molten globule state, which is a collapsed state characterized by high content of secondary structure but less defined tertiary structure (Dobson 1994; Ptitsyn 1995; Redfield 1999; Uversky 2002). In 1981 Dolgikh and co-workers reported that αlactalbumin populates a similar state at low denaturant concentrations (Dolgikh et al. 1981). Since then, this feature has been observed for many proteins using mild denaturing conditions, mutations or removal of co-factors and several studies have indicated similarities between these states and actual folding intermediates (Dolgikh et al. 1984; Ptitsyn et al. 1990; Jennings

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and Wright 1993; Dobson 1994). Molten globule proteins show one or several of the following characteristics: loss of catalytic activity, high degree of secondary but low degree of tertiary structure (seen for instance by far-UV and near-UV CD, respectively), increased dynamics and flexibility (observed by for instance NMR), non-cooperative unfolding, low stability, increased aggregation propensity, and affinity for hydrophobic dyes (such as 8-anilino-1naphtalenesulfonic acid, ANS, figure 5d). Structural investigations of proteins in the molten globule state is of great interest since they can provide information not only about forces that stabilize proteins but also the pathways by which proteins adopt their folded structures.

a

b O

NH H2N

H2N

C NH2 • HCl

c

C NH2

d CH3 H3C

N

O

NH

SO3H

CH3

Figure 5. Chemical structures of (a) guanidine hydrochloride (GuHCl), (b) urea, (c) trimethyl-Namineoxide (TMAO), and (d) 8-anilino-1-naphtalenesulfonic acid (ANS).

16


5 Protein-protein interactions While the driving force for folding essentially relies on the hydrophobic effect, there is a far greater diversity for protein-protein interactions (Jones and Thornton 1996; Stites 1997; Lo Conte et al. 1999). In fact, the conclusion of review articles seem to be that ‘anything is possible’, indicating that there might not be one general mechanism in the same way as for protein stability. An important difference between folding and binding is the reduced influence of the conformational entropy in the binding reaction. This increases the importance of more delicate interactions, which offer a possibility of fine-tuning the process (Tsai et al. 1997; Sheinerman et al. 2000). There are, however, still losses of rotational, translational and internal entropy associated with complex formation. The available structural data of protein-protein complexes is not as extensive as for monomeric proteins. From data available at the time (mostly protease-inhibitor and antibody-antigen structures) Janin and Chothia in 1990 suggested that protein-protein interactions result from burying at least 1200 Å2 total surface area in highly complementary interfaces (Janin and Chothia 1990). This was in good agreement with what had been calculated in the 1970s based on the hydrophobic effect compensating for losses in rotational and translational entropy (Chothia and Janin 1975), and it is still a good approximation of the minimum buried surface area in complexes. The increasing availability of structural data for other types of complexes revealed a far greater diversity for the architecture of protein-protein interfaces than in the core of proteins (Stites 1997; Lo Conte, et al. 1999). At the same time, sensitive microcalorimetry instruments were developed allowing for detailed analysis of the thermodynamics of the interactions (Stites 1997).

5.1 Classification One reason to the variations seen for protein complexes is that they have evolved for different functions and with different requirements of binding affinity and specificity. Several classifications have therefore been used when analysing interaction mechanisms. Most important is the difference between homo- and hetero-oligomeric complexes. The required stability of the isolated subunits in hetero-complexes certainly restricts the possible recognition mechanisms (Jones and Thornton 1996; Tsai et al. 1997). Further classification can be done by comparing the relative sizes of the interacting species (Stites 1997; Liddington 2004) or based

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on the functional origin of the complex (Lo Conte et al. 1999). Tsai and colleagues clustered 1629 protein-protein interfaces into 351 families using a computer-vision based algorithm (Tsai et al. 1996). The results showed, with a few exceptions, that interface similarities were correlated with structural similarities in the subunits. This is consistent with the diversity seen for the interfacial architecture and it also confirms the conservation of interaction surfaces in protein families.

5.2 Structural aspects of the interface Statistics based on structures of hetero-dimeric protein complexes show that complex formation is associated with burying of 1200-4700 Å2 surface area, with a majority of the interfaces having a ‘standard size’ of 1600 ± 400 Å2 (Lo Conte et al. 1999). For homocomplexes, there are examples where more than 9000 Å2 is buried (Jones and Thornton 1996). Homo-complexes also on average have a higher degree of curvature of the interface (Jones and Thornton 1996). Chakrabarti and Janin noted that large interfaces often are divided into two or more patches and that at least one of these is of standard size (Chakrabarti and Janin 2002). There seem to be no specific preferences for the type of secondary structure (Argos 1988; Stites 1997) although residues from several secondary structure elements are involved in most cases (Jones and Thornton 1996). The atomic packing in the interfaces is in general similar to the interior of monomeric proteins (Walls and Sternberg 1992; Lo Conte et al. 1999) even though there are disagreements between different methods to investigate this property. For instance, Jones and Thornton concluded from their gap-index method that antigen-antibody interfaces are less compact than other hetero-complexes (Jones and Thornton 1996). This result could not be confirmed by Lo Conte et al. using Voronoi volumes (Lo Conte et al. 1999). Lo Conte and co-workers further noted the importance of considering water molecules when investigating the packing properties.

5.3 Driving-forces of complex formation The relative importance of different forces for protein-protein interactions is still a matter of debate. The hydrophobicity of protein-protein interfaces seems to be somewhere in-between protein cores and exposed surfaces, with the interfaces of homo-complexes being slightly more hydrophobic than hetero-complexes (Janin et al. 1988; Jones and Thornton 1996). The enrichment of aromatic residues at the interfaces implies hydrophobic contributions, but there

18


is also specific enrichment of arginines indicating that electrostatic interactions are important (Stites 1997; Lo Conte et al. 1999). Further support for the electrostatic forces comes from indications that the desolvation free energy of polar atoms is lower in interfaces than in protein cores (Xu et al. 1997; Sheinerman et al. 2000) and from the effect of long-range electrostatic interactions on the on-rates of complex formation (Schreiber and Fersht 1996). Many, but not all, protein-protein complexes have relatively large ∆Cp° for dissociation, as expected for a process driven by the hydrophobic effect (Stites 1997). However, while the hydrophobic effect is associated with large heat capacity changes, the reverse is not necessarily true. It has been proposed that burial of water molecules upon complex formation could also account for substantial changes in heat capacity (Bhat et al. 1994). Failure to correlate structural based thermodynamic calculations using parameters derived from folding with experimental results (Frisch et al. 1997) further supports the existence of principal differences in the mechanisms of folding and binding.

5.4 The role of individual residues Detailed information about the nature of protein-protein interactions has come from sitedirected mutagenesis studies (Wells 1991; Thorn and Bogan 2001). Two rigorously studied systems are the human growth hormone:receptor complex (Cunningham and Wells 1989; Clackson and Wells 1995; Wells 1996) and the barnase:barstar complex (Schreiber and Fersht 1993; Schreiber et al. 1994; Schreiber and Fersht 1995). From these studies and others it was noted that a few residues, so-called hot spots, contribute considerably more to the binding free energy then others. Certain characteristics are seen for these residues (Bogan and Thorn 1998): i) They are often completely buried and located in the centre of the interface, which led Bogan and Thorn to propose that the lower limit of buried surface area, reported by Janin and Chothia (Janin and Chothia 1990), corresponds to the minimum area needed to create a watertight seal (O-ring) for the hot spots. ii) They interact with other hot spots in the binding partner. iii) Certain amino acid residues (typically Tyr, Trp or Arg) are enriched in hot spots and the very same residues seem to be highly conserved in interfaces (Hu et al. 2000).

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5.5 Affinity and specificity Two central aspects for biomolecular interactions are affinity and specificity. The affinity of a complex is described by the dissociation constant, Kd: Kd = [A] [B] / [AB] = koff / kon

(5)

with [A] and [B] representing the concentrations of the free subunits, [AB] the concentration of the complex and kon and koff the rate constants for association and dissociation, respectively. Kd is related to the free energy of dissociation according to Eq. 1. A wide range of affinities is found for biomolecular complexes in Nature since they have evolved to the ‘right’ affinity, which is not necessarily the highest. For protein-protein interactions, dissociation constants in the nano-picomolar range represent high affinity while the micromolar range denotes intermediate affinity. Antibodies bind antigens with typically nanomolar affinities while protease-inhibitor complexes show slightly stronger binding (Brooijmans et al. 2002). As a reference, the strongest non-covalent bond known is between the protein streptavidin and the small molecule biotin. This interaction has a Kd of 10-15 M (Weber et al. 1989). In addition, proteins with binding functionality must be able to discriminate between possible binding targets. The differences of the binding free energy relative other potential complexes must be sufficiently large for an interaction to be specific. Exactly how large depends on the required specificity but also on the actual concentrations of the competing ligands. The specificity requirements in protein-protein interactions might be one reason for the reduced importance of the hydrophobic effect compared to its role in folding, since hydrophobic interactions are less specific than salt-bridges and hydrogen bonds. As evident from Eq. 5, the affinity can also be expressed in kinetic terms of complex formation and dissociation. The on-rate is limited by diffusion and geometric constraints and is thus sensitive to solution conditions such as viscosity, ionic strength and pH. Typical values for protein-protein associations are 105 - 106 M-1 s-1, but faster reactions have been observed for complexes with strong electrostatic interactions (Sheinerman et al. 2000; Schreiber 2002). The off-rates are determined by the interactions in the formed complex. Mutations in the binding interface therefore mainly affect the off-rate (Cunningham and Wells 1993) even if on-rate effects also have been reported (Jendeberg et al. 1995; Selzer et al. 2000; Spoerner et al. 2001).

20


5.6 Conformational change The structural rearrangements in protein binding processes can be discussed in terms of the ‘lock-and-key’ (Fischer 1894) and induced fit (Koshland 1958) models of enzymatic catalysis, although in a truncated form since no catalytic reaction is considered. Early structural studies showed limited signs of conformational change, which led to the assumption that proteinprotein interactions in general proceed via rigid-body or ‘lock-and-key’ association (Janin and Chothia 1990). As for the interfacial architecture, the increased number of determined structures revealed a more diverse picture with structural changes ranging from reorientation of a few side chains to movement of loops or domains (Lo Conte et al. 1999). There are no strict definitions of how large the rearrangements should be to be classified as induced fit. Stites suggested that it could be discussed in terms of energy rather than in geometric parameters (Stites 1997). If the bound conformation represents a low energy transition it will be populated also in the free state (Kimura et al. 2001), and the binding could be classified as a ‘lock-and-key’ mechanism for that particular conformation. It is obvious that binding of small, elongated peptides will be accompanied with substantial conformational change but there also seems to be a correlation between the interface size and the extent of structural rearrangement (Lo Conte et al. 1999). One reason for this is that large interfaces often involves extended conformations that are not stable in the free protein. Extreme cases of induced fit are seen for the, so-called, intrinsically unstructured proteins (Wright and Dyson 1999; Tompa 2002; Uversky 2002). In the free state they show no or very little regular structure, but accessing the functional state involves binding and coupled folding (Dyson and Wright 2002). This has been demonstrated experimentally by, for instance, Radhakrishnan and co-workers (Radhakrishnan et al. 1997) and in a very interesting paper describing the mutual folding of two intrinsically unstructured domains upon complex formation (Demarest et al. 2002). The functional mechanism of these proteins might seem more similar to folding than protein-protein interaction. However, the reason for the low stability of the free proteins seems to be a lower fraction of hydrophobic residues and a higher degree of polar and charged residues compared to globular proteins (Tompa 2002; Uversky 2002).

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6 Protein engineering Recombinant DNA technology has provided almost unlimited possibilities to manipulate protein molecules. The reasons for doing so are many. In the industry it can, for instance, be to increase the stability of a protein in a chemical process, increase the activity or substrate specificity of an enzyme, designing new drugs or creating specific binding proteins for purification or detection. In academia, manipulating the composition of proteins can provide important information about the fundamental mechanisms of protein folding, stability and interaction. The first successful site-directed mutagenesis was carried out by Michael Smith and colleagues (Hutchison et al. 1978) and the real breakthrough for the method came in 1982, when it was used to reveal the catalytic mechanism of tyrosyl-tRNA synthetase (Winter et al. 1982).

6.1 Rational design Engineering can be performed in more or less rational ways. The efficiency of rational methods relies on the available structural and chemical information of the protein(s) and the understanding of the mechanisms that regulate molecular properties such as stability and affinity. The use of protein engineering to improve proteins was pioneered in the field of enzymatic catalysis. The serine protease subtilisin BPN’ from Bacillus amyloliquefaciens, an important ingredient in washing powder, has been redesigned in terms of substrate specificity (Estell et al. 1986; Rheinnecker et al. 1993; Ballinger et al. 1996) and increased stability (Estell et al. 1985; Pantoliano et al. 1989). Another area where improved performance of certain proteins can be needed is pharmaceutical applications. Stability, solubility and target affinity are all important drug properties that have been optimised through rational design (Marshall et al. 2003). As described in chapter 2, the cores of folded proteins are compact and highly optimised. The possibilities to improve the core packing are thus limited. Instead surface residues have been the targets for many attempts to increase protein stability. Stabilizing the helix dipoles by Nand C-caps (Marshall et al. 2002) or removal of residues with the same charge (Grimsley et al. 1999; Spector et al. 2000; Perl and Schmid 2001) are both approaches based on electrostatic forces at the surface. Destabilization of the unfolded state can be accomplished through decreasing its entropy by introducing proline residues (Muslin et al. 2002) or disulfide linkages

22


(Matsumura et al. 1989; Van Den Burg et al. 1998). Removal of glycine residues will have similar effect (Matthews et al. 1987). Solubility can be improved by reducing surface hydrophobicity (Mosavi and Peng 2003) or by altering the overall charge of the protein, and thereby its isoelectric point (Tan et al. 1998). Binding affinity has also been improved by rational design methods. Using homology and evolutionary data as basis, human thyrotropin was engineered to show 5·104-fold increase in affinity for its receptor (Grossmann et al. 1998). Regulation of ligand binding for an integrin I domain was accomplished by mutations that stabilized either the binding or non-binding conformations (Shimaoka et al. 2000; Shimaoka et al. 2001). A single glycine to arginine mutation in the human growth hormone (hGH) disrupted one of its two receptor-binding sites (Fuh et al. 1992). Since signalling is dependent of receptor dimerization upon hGH binding, this mutant effectively blocks the signalling. In an interesting paper, Kortemme and colleagues describe the redesign of both subunits in a DNase-inhibitor complex to obtain a completely new specificity of the binding (Kortemme et al. 2004). The ultimate test of the understanding of proteins is to design proteins with entirely novel folds or functions. When improving an existing protein it is often sufficient to consider just one or a few aspects. In de novo design, on the other hand, all possible forces and energetic contributions must be taken into account. Hence, it is a problem with many dimensions where the aid of computer algorithms is essential (Dahiyat and Mayo 1997; Ventura and Serrano 2004). Designing helical bundle structures have been a popular goal for many de novo design efforts. The approach has basically been to construct amphiphilic helices and let them aggregate to form a bundle (Baltzer et al. 2001). Substantial work in this field has been done, for instance by DeGrado (Regan and DeGrado 1988) and Hecht (Hecht et al. 1990; Kamtekar et al. 1993). However, many attempts to design proteins this way have resulted in molten globule structures (Olofsson and Baltzer 1996; Aphasizheva et al. 1998; Tahmassebi and Sasaki 1998). For stabilization of a unique native structure it is necessary to introduce some specificity in the helix-helix contacts, which has been demonstrated by for instance Hill and DeGrado (Hill and DeGrado 1998).

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Design of β-sheets has shown to be more difficult, mainly due to aggregation propensity. This could be overcome by introducing charged residues causing repulsion in the aggregate (Baltzer et al. 2001). Furthermore, loop regions connecting the sheets are important determinants of structure (Griffiths-Jones et al. 1999; Espinosa et al. 2001) and including more strands increases the stability (Sharman and Searle 1997; Sharman and Searle 1998). In a fascinating work Regan and co-workers managed to change the β-structure of streptococcal protein G into a four-helix bundle by only changing 50 % of the amino acid sequence (Dalal et al. 1997). Considering that proteins with more than 30 % sequence identity in all known cases have the same structure this is a remarkable achievement.

6.2 Combinatorial methods As an alternative, or in many cases complement, to rational methods combinatorial approaches have been used. These are based on creating a large library of protein variants from which candidates with the desired properties can be selected. The efficiency of the approach relies on the possibility to analyse the whole library in a high-throughput manner. Since DNA technology most often is the basis, the crucial part is to link the phenotype with the corresponding genotype. Methods as phage display, cell surface display, ribosomal display, mRNA-peptide fusions and in vitro compartmentalization have been developed for this purpose (Lin and Cornish 2002). Of special interest for this thesis is phage display. This method was introduced by George Smith in 1985 (Smith 1985) and is based on fusion of the gene of interest to the gene of a surface protein of the phage. The target protein will then be displayed on the phage surface and accessible for selection assays while the corresponding DNA is packed inside the phage particle. Combinatorial approaches are highly suitable to find and improve binding proteins but have also been used to screen for stability (Wei et al. 2003), enzymatic activity (Danielsen et al. 2001) and investigation of amyloid-formation propensity (West et al. 1999).

6.3 Scaffolds for engineered binding proteins For long time immunoglobulins (Ig) were the obvious (and possibly only) choice of highly specific binding proteins. A recent trend is, however, to recruit protein scaffolds for developing novel binding proteins (Nygren and Skerra 2004; Binz and Plückthun 2005). Such

24


constructs might possess several advantages compared to antibodies, including stability, small size and the possibility to produce them by common expression systems or even by chemical synthesis. Early work in this field was focused on small peptide fragments and it resulted in new binders to a number of targets (Smith and Petrenko 1997). However, using stably folded protein units instead of peptide fragments offers benefits such as reduced entropy loss upon binding, higher proteolytic stability, lower sensitivity for incorporation of hydrophobic residues on the binding surface and reduced structural effects of potential fusion proteins (Nygren and Uhlén 1997). As a step towards larger scaffolds, selected peptides were inserted into protein loops, for instance in thioredoxin (Lu et al. 1995; Klevenz et al. 2002) and staphylococcal nuclease (Norman et al. 1999). In theory the evolved binding segments could be cut and pasted into any scaffold suitable for the intended applications. However, this goal has not yet been fulfilled (Nygren and Skerra 2004). As noted in chapter 5, the interacting residues in natural protein-protein complexes are usually spread over several elements of secondary structure. Various proteins, including ankyrins (Binz et al. 2004), cellulose binding domains (Smith et al. 1998; Lehtiö et al. 2000) and a staphylococcal protein A domain (Nord et al. 1997) have been modified to create novel binding activity using neighbouring elements of secondary structure. In antibodies, the binding interface is located to six loops of the complementarity-determining region (CDR). This recognition mechanism has been mimicked using variants of immunoglobulins (Pessi et al. 1993; Martin et al. 1997; Nuttall et al. 2003) or other constructs with similar architecture, such as fibronectin III (Xu et al. 2002) or tendamistat (McConnell and Hoess 1995). The described efforts have proven the concept of protein scaffolds and the variety of novel binders offers the possibility to choose the most suitable construct for each specific application.

6.4 Staphylococcal protein A Central for this thesis is the work performed with a staphylococcal protein A (SPA) domain. Protein A belongs to the bacterial receptin proteins and is exposed on the surface of the bacterium Staphylococcous aureus. The exact biological function for this class of proteins is not known, but they are believed to be involved in recruiting a camouflage for the bacterium (Kronvall and Jönsson 1999). SPA contains five homologous domains (E, D, A, B and C),

Christofer Lendel

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which are all capable of binding immunoglobulins (Ig) from almost all mammals (Moks et al. 1986). In addition, the protein is also expressed with a signal peptide and a cell wall anchoring part (figure 6) (Uhlén et al. 1984). The binding properties vary depending on Ig class and species (Surolia et al. 1982) and it has been shown that the domains are able to interact with both Fc and Fab fragments recruiting different epitopes of the three-helix bundle (figure 7) (Moks et al. 1986; Jansson et al. 1998; Graille et al. 2000). Typical dissociation constants for Fcbinding of SPA domains are 10-8 M (Jendeberg et al. 1995; Starovasnik et al. 1997). XM

+

H3N

COO -

S E

D

A

B

C

Figure 6. Schematic representation of staphylococcal protein A (SPA). The five Ig-binding domains are denoted A-E. S is the signal peptide and XM the cell wall anchoring sequence.

An engineered version of the B domain (denoted the Z domain) was created to improve the resistance to hydroxylamine cleavage (Nilsson et al. 1987). This was accomplished by changing the N28,G29 sequence to N28,A29. The glycine was chosen since the asparagine is involved in Fc binding. In addition Ala1 was replaced by a valine. The Z domain shows retained Fc binding activity but, due to the Gly to Ala mutation, the Fab binding is lost (Jansson et al. 1998). 10 |

20 |

30 40 50 | | | Fab: * ** ** ** * * * * E AQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPK D ADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK A ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPK B ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK C ADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK Z VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK Fc: * *** ** * * ** * | | | | | 10 20 30 40 50 Figure 7. Sequence alignment of the five homologous SPA domains and the Z domain. Completely conserved residues are marked with grey background. Residues involved in Fab and Fc binding are indicated with *.

26


The three-dimensional structures of the B, E and Z domains have been determined by NMR and all show three-helical bundle folds (Gouda et al. 1992; Starovasnik et al. 1996; Tashiro et al. 1997). The high sequence identity between the SPA domains implies that this topology is common for all five domains (figure 7). The backbone conformations of the determined structures are very similar, except for the orientation of helix 1. While the E and Z domains have almost parallel helices, the B domain shows a tilt angle of approximately 30° for the first helix (figure 8). This is surprising, since the B and the Z domains only differ in two positions. The glycine present in the B domain could affect the backbone conformation, but the E domain also has a glycine residue in the same position. To validate the orientation of helix 1, the solution structure of the Z domain was recently refined using residual dipolar couplings restraints (Zheng et al. 2004). The resulting structure is highly similar to the previously determined Z domain structure and also to the E domain structure. Interestingly, Alonso and Daggett noted that the B domain sequence modelled on the Z domain structure (Tashiro et al. 1997) results in lower in vacuo minimized energy, fewer steric clashes and fewer buried unsatisfied hydrogen bonds (Alonso and Daggett 2000). However, molecular dynamics data presented in the same study also indicated fluctuations in the orientation of helix 1 in the B and Z domains but not in the E domain (Alonso and Daggett 2000).

B

E

Z

Figure 8. Structural comparison of the SPA B-, E- and Z domains.

In addition, the co-crystal structure of the B domain and an Fc fragment of human IgG1 has been determined (Deisenhofer 1981). In this structure only helices 1 and 2 are observed in the B domain and it was proposed that Fc binding could be coupled with unwinding of helix 3 (Torigoe et al. 1990a,b). This mechanism would agree with the dimer construct of the Z domain showing substantially increased affinity for Fc, compared to the monomeric equivalent

Christofer Lendel

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(Jendeberg et al. 1995). The Fc fragment presents two identical binding sites for Z but the dimeric construct cannot bind both simultaneously if the three-helix bundle is kept intact. Further data from amide proton exchange (Gouda et al. 1992) and CD (Jendeberg et al. 1996) indicated that neither the monomeric nor the dimeric Z domain lose any structure upon Fc binding. The missing electron density for helix 3 is rather an artefact in the X-ray data than a true structural feature. The binding surface on the Fc fragment is located to the hinge region between the constant domains CH2 and CH3. It was noted by DeLano and co-workers that this epitope possesses intrinsic binding properties, since at least four other proteins recognize it, including a small in vitro evolved peptide (DeLano et al. 2000). The total buried surface area in the B:Fc complex is 1234 Å2 and mainly of non-polar character (see table 1, page 58). 9 residues in Fc and 11 residues in helices 1 and 2 of the B domain participate in the interaction. The energetic contributions from individual residues have been investigated in a mutational study by Jendeberg and colleagues (Cedergren et al. 1993; Jendeberg et al. 1995). The L17D, N28A, I31A and K35A mutations all reduced the Fc affinity of the Z domain, with the largest decrease (∆∆G° = 5.3 kcal mol-1) observed for I31A. It can be noted that the change was attributed to the on-rate rather than of the off-rate, in contrast to what is normally seen for important interface residues (Cunningham and Wells 1993). Braisted and colleagues managed to create a two-helix version of the Z domain capable of Fc binding using a multi-step combinatorial approach (Braisted and Wells 1996). Initially, the removal of the third helix had serious effects on the stability and affinity. However, screening for stability and then Fc binding resulted in a binder with an affinity similar to that of the Z domain. Further stabilization by introduction of a disulfide linkage increased the affinity even more (Starovasnik et al. 1997). Interestingly, several key residues in the interface, including Ile31 mentioned above, were conserved during the selection. The Fab interaction of the SPA domains has been investigated in a co-crystal structure of the D domain and a Fab fragment of human IgM (Graille et al. 2000). The structure revealed that different surfaces are responsible for Fab and Fc binding. Residues Q26, G29, F30, Q32, S33, D36, D37, Q40, N43, E47, and L51 in helices 2 and 3 of the D domain interact with four βstrands in the variable region (VH) of the Fab fragment (figure 7). The total interaction surface is 1220 Å2 and it is predominantly polar. Furthermore, the complex structure also provides an

28


explanation for the lost Fab affinity of the Z domain: The distance between Cα of Gly29 and the target molecule is only 3.5 Å, which is not enough to fit the additional methyl group of an alanine residue. Another interesting feature seen in this structure is the homodimerization of the D domains, involving basically the same residues that are responsible for Fc interaction. It is also worth noticing that the D domain has a three-helix bundle structure with all helices parallel, similar to what is seen for the E and Z domains.

6.5 Affibodies The potency of monomeric and dimeric Z domain constructs as affinity gene fusion partners is well documented (Ståhl and Nygren 1997). This quality results from the high solubility, thermal and proteolytic stability and the absence of cysteine residues. The very same properties make the Z domain a useful scaffold for engineering novel binding proteins. To explore this possibility two combinatorial libraries were created by randomising 9 of the 11 residues involved in Fc binding and 4 additional residues in the same surface (figure 9) (Nord et al. 1995). Amino acid variation in the selected positions

was obtained

oligonucleotides

using

containing

synthetic degenerate

codons. In the first library (Zlib-1) NN(G/T) codons were used, which give access to all amino acids as well as the TAG amber stop codon (Nord et al. 1995). The second library (Zlib-2) was created using (C/A/G)NN codons, which excluded the stop codon and cysteines, but also the aromatic amino acids phenylalanine, tyrosine and tryptophan (Nord et al. 1997). Due to practical limitations the actual size of the libraries were approximately 4·107 variants instead of the theoretical 2013

Figure 9. Z domain backbone trace with the positions randomised in the affibody libraries indicated.

and 1613 for Zlib-1 and Zlib-2 respectively. In a pioneering work, novel binders (so-called affibodies) against three different protein targets (human insulin, human recombinant apolipoprotein A-1 and Thermus aquaticus (Taq) DNA polymerase) were selected using phage display techniques. These binders all showed

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dissociation constants in the micromolar region (Nord et al. 1997), which seems to be typical affinities of first-generation affibodies from these libraries. However, binders with higher affinity have been obtained from larger libraries (Wikman et al. 2004) or by additional randomisation to create second-generation affibodies (Gunneriusson et al. 1999a; Nord et al. 2001). This clearly emphasises the importance of library size. Successful generation of affibodies for several additional targets with dissociation constants as low as 5 nM has been reported (figure 10) (Nord et al. 1997; Hansson et al. 1999; Eklund et al. 2002; Rönnmark et al. 2002; Sandström et al. 2003) and their performance in bioseparation (Nord et al. 2000; Gräslund et al. 2002), detection (Andersson et al. 2003; Renberg et al. 2004) and possible applications in therapy and diagnostics (Sandström et al. 2003; Wikman et al. 2004) have been demonstrated. In addition, the binding properties of these proteins are highly specific as shown by their ability to capture their targets from E. coli cell lysate or periplasmic extract (Eklund et al. 2002), human plasma (Rönnmark et al. 2002) and even in vivo (Orlova et al. 2005; Steffen et al. 2005).

Z domain

10 20 30 40 | | | | VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQ

Zapolipo24:4 ZfVIII26* ZhCD28:5 ZHER2:4** ZIgA1 anti-ZIgA1 ZIgE ZinsulinB6 Zrsv1 ZSPA-1 ZTaq4:1 ZTaqS1-1* anti-ZTaq

·······DKNN·AV··MQ·····AG·IL···I··P····· 3 ········WRM·WV··KL·····YF·RD···M··L····· 0.005 ········ARA·RI··TP·····RE·GG···V··K····· 8.5 ········LRQ·YW··QA·····WT·SR···R··Y····· 0.050 ········TIQ·SQ··RL·····GR·KL···H··L····· 0.5 ········AQT·GV··ME·····TR·LL···Q··R····· 0.9 ········PTA·SL··MM·····VD·VG···G··M····· 0.4 ········AGV·WR··RT·····DE·LM···L··V····· 30 ········ALR·AL··LE·····AH·EL···S··A····· 0.9 ········LSV·GR··VT·····DP·KK···F··W····· 6 ········LGW·TW··FN·····GF·AA···R··R···R· 2 ········LGW·TW··FN·····GV·VK···D··R····· 0.025 ········RVI·IG··MR·····SL·VV···N··R····· 0.7

Kd(µM)

ref. a b c d e,f g f a h g a i g

Figure 10. Alignment of residues 1-40 for a selection of published affibody sequences with the dissociation constants for their targets. *The affibody has gone through an affinity maturation step. **The affibody was selected from a larger library. References: a: (Nord et al. 1997), b: (Nord et al. 2001), c: (Sandström et al. 2003), d: (Wikman et al. 2004), e: (Rönnmark et al. 2002), f: (Gunneriusson et al. 1999b), g: (Eklund et al. 2002), h: (Hansson et al. 1999), and i: (Gunneriusson et al. 1999a).

30


7 Experimental methods to study protein structure As long as protein structure and protein-protein interactions can not be predicted from the amino acid sequence, experimental data will be central for the understanding of proteins. A large number of methods have been used to study a number of structural aspects of proteins, ranging from analysis of amino acid composition to determination of high-resolution structures. I will here briefly describe some of these methods with focus on biophysical techniques and the methods used in the present investigation.

7.1 Size exclusion chromatography In size exclusion chromatography (SEC), proteins are separated due to size and shape by allowing them to pass through a porous gel (Wilson 2000). Small and/or compact molecules experience a larger column volume due to the pores and go through the gel slower than large and/or elongated species. The method is frequently used for purification purposes but it can as well be applied as an analytical tool to obtain information about the compactness or the oligomeric state of a protein. Quantitative measurements of the binding constant in low affinity complexes have been deduced from size exclusion chromatography elusion profiles (Hansson et al. 2001). Alternative methods that can provide similar data are analytical ultracentrifugation and light scattering experiments.

7.2 Isothermal titration calorimetry A highly sensitive method for measuring the thermodynamics of binding reactions is isothermal titration calorimetry (ITC) (Pierce et al. 1999; Velazquez-Campoy et al. 2004). Several aliquots of ligand solution are titrated into a protein solution and the energy required for keeping a constant temperature difference between the reaction cell and a reference cell is measured. From the data, the enthalpy of binding, the association (or dissociation) constant and the stoichiometry of the binding can be obtained. Using the measured values of ∆H° and ∆G° (from Ka), ∆S° can be calculated using Eq. 2. For a complete thermodynamic profile, ∆Cp° can be obtained from fitting a line to ∆H° measured at several temperatures. Due to the sensitivity of the measurements it is important to make the appropriate control experiments to check the effect of pH, ionic strength etc. (Stites 1997; Velazquez-Campoy et al. 2004).

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7.3 UV-VIS spectroscopy UV-VIS spectroscopy provides a fast and useful method for investigating protein structure due to the delocalised π electrons of the peptide bond and the aromatic side chains (Fersht 1999). Absorbance and fluorescence measurements can provide similar information, although fluorescence is more sensitive since it is measured relative to a dark background orthogonal to the light source. The UV absorption spectra of proteins show two maxima: 190-210 nm, where the peptide bond absorbs and around 280 nm, with the main contribution from tryptophan and tyrosine side chains. The tryptophan fluorescence in the 330-340 nm region is a common method to monitor protein unfolding, since the emission spectrum and intensity are highly sensitive to the surroundings. Similar effects can also be seen for some external probes, such as ANS. Binding of this dye in hydrophobic pockets in partially folded states (molten globules) results in a substantial change in the fluorescence spectrum. Also decreased fluorescence (quenching) can be used to extract structural data, for instance when the ligand binding is accompanied with quenching of a fluorophore. If two different probes for which the emission spectrum of the first overlaps with the excitation spectrum of the second one are present, a direct transfer of energy between these probes can take place (Förster 1948). The phenomenon is called fluorescence resonance energy transfer (FRET) and provides a method to detect spatial closeness between the probes (Miller 2005).

7.4 Circular dichroism spectroscopy A well-established method for investigating secondary and tertiary structure of proteins is circular dichroism (CD) spectroscopy. The method is based on the difference in absorption of right- and left-handed circular polarized light observed for optically active molecules (Woody 1996). CD originating from the chiral properties of the peptide backbone (see chapter 2), can be measured in the far-UV region (170-250 nm) and is sensitive to the type of secondary structure (figure 11). As mentioned above, aromatic side chains absorb light in the near-UV region. These are normally not optically active, but can display such properties when placed in an asymmetric environment, as in the core of a folded protein. The CD signal in this region (270-300 nm) is thus sensitive to tertiary structure. Several empirical methods for estimating the secondary structure composition of proteins from their CD spectra have been developed (Venyaminov and Yang 1996). The results are not always very accurate, especially not for proteins with mixed secondary structure. However, for exclusively helical proteins the methods

32


are often reliable and have therefore provided an important tool for investigating the mechanisms of helix-coil transition (Kallenbach et al. 1996).

CD (a.u.)

β - sheet

Turn

α - helix Random coil 180

200

220

240

260

Wavelength (nm)

Figure 11. Typical far-UV CD spectra for different types of secondary structure.

7.5 Nuclear magnetic resonance spectroscopy Nuclear magnetic resonance spectroscopy (NMR) provides a powerful tool for structural studies of proteins and plays a central role in this thesis. The foundations were built in the late 1960s and during the 1970s by the invention of Fourier transform- and two-dimensional NMR techniques. In the 1980s Ernst and Wüthrich developed the methodology for using NMR in structural studies of biomolecules (Ernst et al. 1987; Wüthrich 1986). NMR spectroscopy is based on transitions between magnetic energy levels of the atomic nuclear spin (Cavanagh et al. 1996). These transitions are in the radiofrequency (MHz) region and can be observed when the sample is placed in a strong magnetic field (typically 10-20 T). Modulations of the transition energies by the surrounding offer many probes of structural and chemical properties. Small deviations in the local magnetic field experienced by each nucleus give rise to the so-called chemical shift. The shifts are typically parts per million (ppm) of the resonance frequency and they report on the chemical nature of the immediate surroundings. Scalar couplings (also denoted J-couplings) allow specific nuclear interactions through the connecting chemical bonds. These types of connections can be used to explore the local structure of proteins. Furthermore, the nuclear spins interact through dipolar couplings, which are not restricted to chemical bonds. This means that couplings will be seen for atoms with

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large separation in sequence but close in space. In isotropic media these interactions are averaged out but can be reintroduced by introducing anisotropy in the solution (Bax 2003). However, cross-relaxation mediated by dipolar interactions - the important nuclear Overhauser effect (NOE) - can still be measured in an isotropic solution (Cavanagh et al. 1996). The distance dependence of these interactions is, as for dipoles, r-6, which makes the NOE an efficient probe of atomic distances. A typical protein structure determination by NMR proceeds in four steps: i) sample preparation, ii) assignment of the resonance frequencies (chemical shifts) for each atom, iii) identification of NOE correlations and conversion of these to distance restraints and iv) structure calculations using the obtained information. Since NMR is a relatively insensitive method, a protein sample must typically contain 1-2 mM protein. Recent advances with cryogenic detection probes have, however, made it possible to use much lower concentrations (Moskau et al. 2003). The protein must also be stable for several weeks, since acquisition of a complete data set is time consuming. And most important: the protein cannot be too large since this results in increased spectral overlap and rapid relaxation of the signal (line broadening) caused by slow molecular motions. The size limitation for standard techniques is approximately 40 kDa. Even if not required, it is often practical to use isotopic (13C, 15N, 2H) labeling. Provided the sample, J-coupling correlations in local structural elements are used to identify all (or as many as possible) atomic resonance frequencies in the protein. A smorgasbord of experiments that correlate different atoms in the protein is available (Cavanagh et al. 1996; Sattler et al. 1999). With each atom assigned to a resonance frequency, NOE correlations can be identified in the nuclear Overhauser correlation spectroscopy (NOESY)-type spectra and distance information retrieved. In theory it is possible to calibrate peak intensities with distances, but due to several reasons, e.g. angular dependence, spectral noise, resonance overlap, dynamics and spin diffusion, this is normally not done. Instead the NOE correlations are divided into a few classes, for instance weak, medium and strong NOEs corresponding long, medium and short distance restraints, respectively. The distance restraints can be complemented with other types of NMR derived data. For instance dihedral angles obtained from measuring J-couplings (Cavanagh et al. 1996) or residual dipolar couplings from slightly aligned systems (Bax 2003). The structures are then calculated using distance geometry algorithms or simulated annealing protocols. Since the system is underdetermined, many

34


structures can be consistent with the restraints. The final NMR structure is therefore represented by an ensemble of structures.

Bond vibrations

Rotational diffusion

Concerted atomic motion

Folding

Rotation of surface side chains

Allosteric transitions

Disordered backbone motions

Rotation of buried sidechains

Relative motions of domains Local unfolding

-12

-9

Heteronuclear R1, R2, NOE etc.

-6

-3

R2ex, R1rho, etc

Unfolding

0

3

log(time/sec)

Saturation transfer Exchange out/quenching

Figure 12. Illustration of the time scales for a number of dynamic events in proteins (above the axis) and the accessible time scales of some NMR methods (below the axis). Figure kindly provided by T. Härd.

NMR also provides means to investigate dynamic events on different time scales. Figure 12 illustrates the time scales for different types of motions in biomolecules and some NMR experiments that can be used to detect the motions. Several techniques rely on magnetic relaxation mechanisms (Cavanagh et al. 1996), which are highly sensitive to the correlation times of molecular motions. R1 (longitudinal- or spin-lattice relaxation), R2 (transverse- or spinspin relaxation) and heteronuclear steady-state NOE experiments detect fast motions on the pico to nano seconds time scales (Palmer 2001). In addition, the R2 rates can contain contributions from chemical exchange. This effect is most pronounced when the exchange rate is comparable with the chemical shift difference between the exchanging states, typically on micro to milliseconds time scales. Rotating frame R1 (R1ρ) experiments is another method to detected these motions (Akke 2002). Slow motions (slower than the seconds time scale) can be detected from the exchange rates of hydrogen atoms in 100 % D2O (Redfield 2004). The exchange rates are correlated with the molecular motions since structurally protected hydrogen atoms have slower exchange rates than if they were completely solvent exposed. Fast hydrogen exchange can be monitored as decrease in signal intensities in an experiment preceded by saturation of the water resonance frequency, a so-called saturation transfer experiment (Forsén and Hoffman 1963).

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7.6 Other methods to determine 3D structures Even though NMR provides an invaluable tool for structural studies of proteins, the most established method for determining high-resolution structures of biomolecules is crystallographic X-ray diffraction (Branden and Tooze 1999). The X-rays are scattered by the electron clouds in the studied molecules with the crystal lattice functioning as a grating, giving constructive interference and amplification. From the diffraction pattern an electron density map is calculated which is used to build a model of the investigated molecule. The major obstacles, besides getting the crystals, are to obtain the phases of the diffraction pattern. This can be done by recording data for several heavy metal derivates, using seleno-methionine labeling or by molecular replacement if a similar structure is known (Branden and Tooze 1999). Another method that becomes increasingly important is electron microscopy (EM). This method has earlier been restricted to obtain low resolution structures but recent development of cryogenic EM techniques have substantially improved its performance (Orlova and Saibil 2004; Subramaniam and Milne 2004). Unlike X-rays, electrons can be focused and both the diffraction pattern and a direct image can thus be obtained. The main difficulty is to obtain sufficient sensitivity, which requires high electron doses, without damaging the sample. This can be achieved by negative metal staining of the specimen or, more efficiently for biomolecules, by collecting the data at cryogenic temperatures. Further improvement of the signal-to-noise ratio is obtained if the molecules display intrinsic or crystalline symmetry. Statistics from the protein data bank (PDB) (Berman et al. 2000) shows that NMR has only contributed to 15 % of the deposited structures. X-ray crystallography is the oldest method and also the fastest and giving highest precision when the crystals have been obtained. But the requirement of high quality crystals is also its major limitation. Even though methods for obtaining crystals have been developed into today’s large scale screening methods, some proteins still do not easily form crystals. The possibilities to study highly dynamic states are also limited and the structure of the protein surface might be affected by the crystal lattice. NMR spectroscopy does not require crystals, but instead there is a size limitation. Although the introduction of the so-called TROSY method (Pervushin 2000) has greatly facilitated investigations of large proteins, size still matters. Another limitation is the time consumption, even though progress has been made in automation of data collection and analysis during the recent years (Güntert 2004; Liu et al. 2005). The major advantage of NMR is, however, the possibility to detect and quantify dynamic events. This makes it a technique suitable not only

36


for studying partially folded states but also to quickly assess the state of proteins in screening experiments (Woestenenk et al. 2003).

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37

Present investigation The work presented in this thesis concerns the molecular mechanisms of stability, affinity and specificity in affibody proteins. These mechanisms have not been studied to any greater extent for this kind of artificial binding proteins and the available structural data of such complexes is still very limited (Binz and Plückthun 2005). The NMR structures presented in this thesis are not only the very first structures of affibody proteins; they also represent a pioneering work in structural characterization of novel binding proteins. Hopefully an increased understanding of these proteins will improve scaffold selection, library construction and selection procedures. There is also a more general aspect of this study. As outlined in the introduction, there is a limited understanding of the basic mechanisms of protein-protein interaction and protein stability. Affibodies and affibody:affibody complexes represent very interesting model systems for studying such mechanisms. Their small size makes them suitable for NMR methods, which is only the case for a limited number of protein-protein complexes. Furthermore, anti-idiotypic affibody complexes offer the possibility to investigate several interaction interfaces without having to analyse completely new proteins and the homology of the proteins makes it easier to identify the causes of any differences in their behaviour. In addition, the specific side chain substitutions in the affibody constructs are likely to affect the stability of the basic scaffold to various extent. Investigating the free proteins will thus give information about both the general aspects of protein stability and how stability influences the binding process. For a deep understanding of a chemical or biological system the experimental data must be as complete as possible. The work presented here is therefore not limited to the structure of the protein-protein complexes. Even if such data has provided many clues about the mechanisms of protein-protein interactions (e.g. Lo Conte et al. 1999), it does not give the full picture. The present investigation, in addition to the structural data of the complexes, contain structure determination and biophysical characterization of the unbound proteins, quantification of the dynamic events on different time scales and investigation of the thermodynamics of folding and complex formation. Taken together, these data provide a very detailed description of the system under investigation.

38


8 Structural investigation of the Z:ZSPA-1 complex (I, II) In a project with the objectives to obtain Z domain-based affinity protein pairs Eklund and coworkers isolated three new affibodies: ZSPA-1, anti-ZTaq and anti-ZIgA (Eklund et al. 2002). These were selected from a mixture of the Zlib-1 and Zlib-2 libraries. ZSPA-1 was selected using a fivedomain SPA construct as panning target and a competitive elution protocol with IgG, capable of competing with library members recognizing both the Fc and Fab binding sites. Surface plasmon resonance studies showed that ZSPA-1 binds all SPA domains with dissociation constants in the range of 2-6.5 µM. There were several reasons for choosing this complex as starting point for the investigation: i) the fascinating fact that ZSPA-1 represents a binder to its own ancestor; ii) the available structural data for the free and Fc-bound Z (B) domain; iii) the possibility to use the Z domain as comparative standard when characterizing the free affibody and iv) the potential use of ZSPA-1 as affinity partner in biotechnological applications using SPA. The proteins were produced in E. coli, using 13C,15N isotopically enriched medium and purified as described in (I). The isotope labeling increases the speed and robustness of the NMR analysis and provides additional structural restraints extracted from the chemical shifts of the carbon and nitrogen nuclei. In this study, the labeling strategy was necessary due to high similarity of the proteins in the complex. Without combining one labeled and one unlabeled subunit in the NMR samples, it would not have been possible to resolve the resonances from the two proteins. Furthermore, this approach allowed for identification of intermolecular contacts using NOESY experiments with special isotope filtering (Vuister et al. 1994). The pH was chosen to be 5.6 due to reduced sample solubility at higher pH and the temperature was set to 30 °C based on the appearance of the NMR spectrum. The NMR assignments was done in a standard stepwise manner, starting with the backbone, followed by the side chains and finally the NOE connectivities. The procedure was straightforward due to the small size of the proteins, the labeling strategy and the available chemical shifts of the free Z domain (Tashiro et al. 1997). As presented in (I), an essentially complete assignment of the

1H,

13C

and

15N

resonances were obtained. Using the

conformational dependence of J-couplings and NOEs, a number of methylene Hβ protons could be stereospecifically assigned from

15N-TOCSY

and

15N-NOESY

experiments with

Christofer Lendel

39

short mixing times (Clore et al. 1991). At the same time χ1 dihedral angle restraints were obtained for these residues. The stereochemistry of the valine and leucine methyl groups were elucidated using separately produced protein with partial 13C-labelling (Neri et al. 1989). Due to the stereoselectivity of the pathways for valine and leucine biosynthesis, the incorporation of a 13C

atom in the position adjacent to the methyl group only occurs for the pro-R variant. The

stereochemistry of the methyl groups can thus be resolved, for instance from the relative phases of the resonances in a constant-time 13C-HSQC. The structure of the complex was determined from a total of 2449 NMR derived restraints, corresponding to 21.1 restraints per residue (II). The r.m.s.d. for all heavy atoms in the ordered parts (residues 8-56 in both subunits) is 0.70 ± 0.07 Å. Both proteins use helices 1 and 2 for binding, which for the Z domain means the Fc binding surface. The fact that ZSPA-1 recognizes the Fc-binding surface of the SPA domains is probably due to the employed competitive elution. Another reason could be a counter-selection for binders to regions leading to selfrecognition, which might inhibit the selection procedure at some stage. The interface buries a total surface area of 1632 Å2, which is predominantly non-polar (64 %). The hydrophobic parts form a central core of the interface while the polar interactions occur at the edges. Other relevant structural data includes 10 intermolecular hydrogen bonds, one salt-bridge and a total of 186 interface atoms belonging to 32 residues (figure 13). 10 out of 13 of the randomized positions in the ZSPA-1 affibody are directly involved in binding. However, Asp24 only uses the backbone carbonyl for contacts, which makes the actual amino acid type less important. Pro25 seems to have an indirect function by fixating the backbone in a position suitable for Asp24 to accept a hydrogen bond. Residue substitutions without any apparent function are thus N11V, H18T and E24D. However, ITC data for a ZSPA-1(V11N) mutant indicates reduced affinity with an asparagine in this position (III). The nature of this residue is thus important, but it is not possible to conclude if the valine is especially favourable or if the asparagine is unfavourable. An X-ray structure of the complex was independently determined by Högbom and co-workers (Högbom et al. 2003). A comparison of this structure and the NMR structure shows that the backbone conformations and the core packing are almost identical. There are, however, some minor differences mainly involving polar side chains and electrostatic interactions. For instance, the acceptor of the Tyr14 (Z) hydrogen bond is, in the NMR structure, the backbone

40


a

Y14

H18

L17 R27

N11

K7

E24

F13

N28

I31 L34 Q9

Q10 Q32 K35 D36

*D24 *P25

b

*K28

*K27

*V17

*F32

I31

*R14 *G13

L34 *W35

*S10

*L9 P3

K7 N6

Figure 13. The Z:ZSPA-1 interface. (a) Helices 1 and 2 of the Z domain upon a surface representation of the ZSPA-1 affibody. The side chains of the interacting residues are indicated. (b) Helices 1 and 2 of ZSPA-1 upon a surface representation of the Z domain. Residues in the randomised positions are coloured in yellow. Randomised positions that are not involved in intermolecular contacts are marked with stars.

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41

carbonyl of Asp24 (ZSPA-1) while the X-ray structure indicates a water-mediated hydrogen bond to the Asp24 (ZSPA-1) side chain. Garbuzynskiy and colleagues recently presented a comparative study of X-ray and NMR structures solved for the same proteins (Garbuzynskiy et al. 2005). They noted that, even if the two methods in general give very similar structures, there are certain systematic differences involving atomic packing and hydrogen bonding. These differences are similar to what is seen for refined and re-refined NMR structures, indicating that the reason might be the refinement protocols rather than the experimental data. Considering these findings, together with the high sulphate concentration in which the crystals were obtained, the differences seen for the Z:ZSPA-1 complex are insignificantly small. 40

2.0

1.0

Rex

30

R2 (s-1)

0.0 0.0

20

0.2

(Dw)

2

0.4

10

0

0

10

20

30 Residue

40

50

60

Figure 14. Transverse relaxation rates (R2) of the Z domain in the Z:ZSPA-1 complex measured at 30 °C on a 600 MHz Bruker Avance NMR spectrometer. The sample composition was the same as described in (I and II) CPMG-delay τ = 2.0 ms. Inset: Rex (obtained from CPMG relaxation dispersion measurements (Loria et al. 1999) with the delay τ = 0.5, 0.75, 1.0, 1.5, 2.0 and 2.5 ms) plotted against the squared chemical shift difference defined as (∆ω)2 = ((∆ω15N)/5)2 + (∆ω1H)2 .

In addition, NMR was used to investigate the dynamics of the subunits in the complex (C. Lendel and P. Allard, unpublished data). No extraordinary dynamics were observed, except for a few residues in helix 1 of the Z domain that showed increased 15N transverse relaxation rates (R2), possibly from chemical exchange (Rex) contributions (figure 14). The exchange contributions were quantified using

15N

Carr-Purcell-Meiboom-Gill (CPMG) relaxation

dispersion experiments (Loria et al. 1999). The Rex terms are proportional to the square of the chemical shift difference between the exchanging states (Loria et al. 1999). There were, however, no obvious correlation between the measured Rex values and the squared chemical shift differences between the free and complex bound state (figure 14). The dynamics could

42


thus not represent the global on-off rates of the complex formation. More likely it results from local fluctuations in the complex.

a

Z:ZSPA-1

b

B:Fc

c

Z : ZSPA-1 B : Fc Free Z

Figure 15. (a,b) Comparison of the interfaces in the Z:ZSPA-1 complex (a) and the B:Fc complex (b). (c) Induced fit of Tyr14 and His18 upon complex formation

The Z domain uses essentially the same residues for ZSPA-1 binding as the B domain uses for the Fc interaction (figure 15) (Deisenhofer 1981). Interestingly, some of these (e.g. Tyr14 and His18) seem to undergo similar rearrangements upon binding to ZSPA-1 as observed in the B:Fc-complex. Even more fascinating are the topological similarities of the interaction surfaces of the ZSPA-1 affibody and the Fc fragment. The ability of anti-idiotypic antibodies to mimic the corresponding antigen was postulated by Jerne in 1974 (Jerne 1974), but there is very limited structural data confirming this on a molecular level (Braden et al. 1996; Vogel et al. 2000). Considering this, the finding that the small helical affibody mimics the large, and structurally far more complicated, Fc fragment was quite unexpected. A similar observation, although not this extensive, has later been seen for another in vitro evolved binding protein. The lipocalin variant DigA16, selected by phage-display to bind to the steroid digoxigenin, showed almost identical configuration of three aromatic side chains as in the complex structure of an antibody binding the same steroid (Korndörfer et al. 2003). These examples indicate that in vitro selection methods are capable of mimicking Nature’s solutions for specific molecular recognition.

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9 Biophysical characterization of the ZSPA-1 affibody (II, III, IV) The first indications that the free ZSPA-1 is more dynamic than the parental Z domain came from the 15N-HSQC NMR spectrum. This is an efficient probe to access the state of a protein if it is available with 15N labelling. Significant for a structured protein are narrow resonances with a good dispersion and deviations from these features indicate the presence of dynamics and/or unstructured parts. The

15N-HSQC

spectrum for ZSPA-1 is shown in figure 16.

Compared to the complexed form of ZSPA-1 or the free Z domain it displays broader resonances, some of them even so broad that they have completely disappeared. Further characterization using CD revealed that ZSPA-1 nevertheless has substantial helix content, but not as high as the Z domain (figure 17a). Moreover, ZSPA-1 has a decreased melting temperature and a less cooperative melting transition compared to the Z domain (figure 17b). These observations indicated that the free affibody might be a molten globule and it was therefore tested for ANS binding. As described in chapters 4.5 and 7.3, binding of ANS to hydrophobic pockets or partially folded states, such as molten globules, results in an enhanced fluorescence. Compared to the free Z domain or the Z:ZSPA-1 complex, the addition of free ZSPA-1 to an ANS solution substantially increases the fluorescence (figure 17c). Hence, it was concluded that

a

b

c

15N

chemical shift

ZSPA-1 is a molten globule.

1H

chemical shift

Figure 16. 15N-HSQC spectrum of the ZSPA-1 affibody (a) without the Z domain, and with (b) 50 % and (c) 100 % relative molar concentration of the Z domain added. Figure kindly provided by E. Wahlberg.

44

Fluorescence intensity (arbitrary units)

Mean residue ellipticity (deg cm2 dmol-1)

Mean residue ellipticity (deg cm2 dmol-1)


a 0

500 µM 400 µM 200 µM

-10000

50 µM

-20000

25 µM

-30000

ZSPA-1

Z domain (25 µM)

200

220

240

Wavelength (nm)

ZSPA-1

Z + ZSPA-1 Z free ANS

520

b ZSPA-1

-20000

Z domain

-30000 20

40 60 80 Temperature (°C)

Figure 17. (a) Far-UV CD spectrum of the Z domain and the ZSPA-1 affibody at several concentrations. (b) Thermal melting of ZSPA-1 and the Z domain monitored with CD at 208 nm. (c) ANS fluorescence in buffer and with equimolar amounts of the Z domain, ZSPA-1 and Z:ZSPA-1 complex added.

c

420

-10000

620

Wavelenght (nm)

In addition, there are signs of some kind of self-association at higher (millimolar) concentrations. This was first seen from size exclusion chromatography experiments, which show shorter retention times than expected and asymmetric elution, indicating an equilibrium between species of different size (figure 18). The concentration dependence of the CD spectrum (II) further indicates that aggregation is accompanied by changes in secondary structure content (figure 17a).

Absorbance

Figure 18. Size exclusion chromatography (SEC) elution profiles of the Z domain (solid squares), the Z domain + 13.7 kDa ribonuclease A (open squares), ZSPA-1 loaded at 2 mM (solid circles) and ZSPA-1 loaded at 0.1 mM (open circles).

40

45

50

55

60

Elution volume (ml)

65

70

Christofer Lendel

45

Paper III presents an extensive investigation of the molecular properties of the molten globule state. The dynamics causing the line broadening in the 15N-HSQC spectrum could originate from several sources: the unfolding equilibrium, the self-association and intrinsic motions in the molten globule state are all reasonable explanations. Several approaches were tested to improve the appearance of the NMR spectrum. The combination of low protein concentration (50 µM) and decreased temperature (20 °C or below) resulted in spectral improvements. These conditions are, however not ideal for quantitative NMR measurements. Instead, the effect of different stabilizing agents was investigated. Addition of 1-1.6 M TMAO was found to greatly improve the appearance of the NMR spectrum and allowed for characterization of the structure and the dynamics on different time scales. The majority of the backbone resonances were assigned for ZSPA-1 in TMAO. This allowed for identification of regions of secondary structure by secondary chemical shifts (Wishart and Sykes 1994a; Wishart and Sykes 1994b) (figure 19a). The data is consistent with all three helices being present, which means that the reduced helicity observed by CD is due to transient unfolding rather than missing helices. This conclusion was confirmed by the presence of characteristic NOE-patterns for helical structure. Further investigation of the NOESY spectrum led to identification of several long-range NOEs consistent with the tertiary structure seen for ZSPA-1 in the complex. These observations are not per se evidence for a folded protein, they only mean that these contacts are present in the ensemble of inter-converting structures. Hence, both the secondary and tertiary structures are native-like, but to understand the nature of the molten globule state quantification of the dynamics was needed. The 15N backbone dynamics was investigated on different time scales. The steady-state 1H-15N NOE experiment, which report on fast (ps-ns) motions, did not reveal anything extraordinary. The transverse relaxation rates, R2, have overall values that are higher than expected (figure 19b). These rates are sensitive to the rotational correlation time and the increased values are probably a result of the self-association mentioned above. Some residues show even higher values due to additional contributions from chemical exchange on an intermediate (µs-ms) time scale. Such motions are also responsible for the spectral line broadening. To quantify the line broadening the peak heights were compared with the peak volumes in the

15N-HSQC

(figure 19c). These data clearly show that the line broadening is most extensive in helices 1 and 2 and they are consistent with the fact that the unassigned backbone atoms are located in the same regions. Successive removal of TMAO from the sample increased the line broadening for the whole protein due to contributions from the overall unfolding equilibrium. Finally,

46 Ca secondary shift (ppm)

Molecular principles of protein stability and protein-protein interactions 6

A

4 2 0 -2 40

10

20

30

40

50

10

20

30

40

50

10

20

30

40

50

10

20

30

40

50

B

R2 (s-1)

30 20 10

Peak intensity:integral

0

0.09 0.07 0.05 0.03 0.01 1.2 1.0

I / I0

C

D

0.8 0.6 0.4 0.2 0

Residue Figure 19. NMR data for ZSPA-1. (a) Secondary Cα chemical shifts (Wishart and Sykes 1994a) of ZSPA-1 in 1.6 M TMAO. The bars indicate the positions of the helices in the Z-bound ZSPA-1 structure (II). (b) Transverse relaxation rates (R2) of ZSPA-1 in 1.6 M TMAO. (c) Peak intensity:integral ratios of resonances in the 15N-HSQC spectrum of ZSPA-1 with (open diamonds) and without (solid circles) 1 M TMAO. (d) Saturation transfer protection of the Z domain (solid squares) and ZSPA-1 with (open diamonds) and without (solid circles) 1 M TMAO. Typical error bars for data in (c) and (d) are shown in the upper right corners.

Christofer Lendel

47

saturation transfer experiments report about dynamic events on a slow (ms-s) time scale (figure 19d). These measurements are also in agreement with an increased mobility in helices 1 and 2. To summarize the dynamics: the residues in helices 1 and 2 undergo structural interconversions on intermediate-to-slow time scales. Interestingly, there are experimental (Bai et al. 1997) and simulation (Alonso and Daggett 2000) data for the B domain of SPA indicating a higher intrinsic stability of helix 3 compared to helices 1 and 2. One explanation for this could be that the protein has sacrificed some stability in order to optimise the Fc binding. If this is the case, this region might be more sensitive to mutations than the rest of the protein. Redfield and colleagues demonstrated that the four-helix bundle interleukin-4 at low pH adopts a state showing some molten globule characteristics although other properties indicate a highly ordered state (Redfield et al. 1994). Characterization of the dynamics using NMR showed substantially decreased order parameters for certain regions, indicating local unfolding. The core, however, remained stable. This local unfolding resulted in increased ANS binding and changes in near-UV CD. In ZSPA-1 the concept of local fluctuations seem to be valid even though they occur on another time scale. Due to the small size of the protein, the fluctuations also lead to disruption of the core. Olofsson and Baltzer investigated the structural properties of the de novo designed helix-loophelix dimer SA-42 using NMR and CD (Olofsson and Baltzer 1996). In water solution this protein shows molten globule behaviour with increased NMR line broadening. In analogy with our work they used a co-solvent to improve the spectral appearance. The primary effect of the used trifluoroethanol (TFE) is increased lifetimes of the hydrogen bonds, resulting in induced helical structure but also disruption of the tertiary and quarternary packing, which was evident from the lack of long-range NOEs at high TFE concentrations. However, at low TFE concentrations the structural properties was similar to those in water, and information about helicity and inter-helical packing could be obtained. Even if this study shows many similarities with the present investigation there are a two important differences: i) TMAO does not, according to the presented data, affect the structural properties of the molten globule state, but rather reduces the influence of other processes, such as the unfolding. ii) The molten globule character of the SA-42 is related to the core packing, possibly due to too high helix propensity

48


of the sequence. The folding problems of ZSPA-1 are, on the contrary, caused by the surface exposed residues and too low helix propensity. The reduced stability of ZSPA-1, compared to the Z domain, as seen in the thermal melting, is confirmed by unfolding experiments using GuHCl. van’t Hoff analysis of the thermal unfolding data presented in (IV) gives further clues about the nature of the free affibody. The ∆G°unfold is seriously reduced compared to the Z domain, an effect that seems to be mainly entropic. Since the molten globule should have higher conformational entropy than the folded Z domain this entropy difference must originate from the solvation entropy, which is in agreement with the hydrophobic effect being less important due to reduced packing quality. Further evidence for this comes from the difference in ∆Cp°,unfold seen for the ZSPA-1 and the Z domain. As mentioned in chapter 3, the hydrophobic effect is closely related to a large value of ∆Cp°. The reduced ∆Cp° value seen for ZSPA-1 unfolding is thus consistent with the exposure of hydrophobic surface in the molten globule state. Native = MG

Unfolded High temp GuHCl

Z bin ding

TMAO

H ig

Folded

h

co

nc

Aggregated

Figure 20. Schematic illustration of the accessible states for the ZSPA-1 affibody. Figure adopted from T. Härd.

Compiling the structural information of the free ZSPA-1 affibody results in the scheme illustrated in figure 20. The molten globule state has native-like secondary and tertiary structures. Increased dynamics is, however, observed for helices 1 and 2, which leads to poor core packing and reduced stability. The low energy difference between the native (molten

Christofer Lendel

49

globule) and unfolded states results in a relatively high population of the unfolded state. The unfolding equilibrium is affected by high temperature or denaturant concentration in favour of the unfolded state. TMAO, on the other hand, drives this equilibrium in the reverse direction, but it does not induce well-defined packing in the molten globule. The compact, Z domainlike, state is only energetically stable when bound to the target Z domain. Finally, increased protein concentration promotes an aggregated state. The nature of this state has not been further investigated but SEC data suggests that the dissociation constant for the selfassociation is in the millimolar region. This process should thus not compete with the target binding. Four mutants were constructed in an attempt to answer the question why ZSPA-1 shows this behaviour. Three different amino acid residues in ZSPA-1 were replaced with the corresponding ones from the Z domain to test if the reason for the molten globule characteristics could be the exposure of hydrophobic surface. In ZSPA-1, 64 % of the exposed surface area is non-polar (as judged by the complex structure), while it is 55 % for the Z domain. An average value for globular proteins is 57 % (Miller et al. 1987). Based on this assumption and the fact that it is not directly involved in the binding, Val11 seemed to be a suitable target for mutation. In addition, the results from the dynamics investigation indicated that Phe32 and Trp35 could be key-residues for stability. The investigated mutants were thus V11N, F32Q, W35K and a F32Q/W35K double mutant, which all should reduce the hydrophobicity of the surface. The mutants were characterized by a set of biophysical methods, including CD, thermal- and chemical denaturation,

15N-HSQC,

ANS-binding, SEC and investigation of the Z domain

binding using ITC. The results showed that at least the F32Q mutation improved the behaviour of ZSPA-1, but the differences were in many aspects small. Hence, the hydrophobicity of the exposed surface could not be the only explanation for the reduced stability. A better correlation was observed if the helix propensity, determined from the consensus secondary structure prediction method (Combet et al. 2000), was taken into account. The effect of surface residues on protein stability is far more complicated than the role of the hydrophobic core. The strengths of different forces become difficult to evaluate at the waterprotein interface, especially since the residues will have approximately the same surroundings in the folded and unfolded states. One important issue is optimisation of charge-charge interactions (Grimsley et al. 1999; Spector et al. 2000; Sanchez-Ruiz and Makhatadze 2001; Perl and Schmid 2001), which could be the reason for the reduced stability of the W35K mutant, as

50


discussed in (III). In an extensive undertaking Funahashi and co-workers studied the effect of surface exposed hydrophobic residues on stability (Funahashi et al. 2000). Three positions, in a helix, a β-sheet and a loop, in human lysozyme were changed from Val to Gly, Ala, Leu, Ile, Met and Phe. X-ray structures and thermodynamic data for unfolding were acquired for all mutants. The effect of specific mutations was found to be highly dependent on position, indicating that intrinsic secondary structure propensities are important. Using a theoretical deconvolution of the energetic contributions they identified the hydrophobic effect and structural changes of water hydration as the most important contributors to the changes in stability. The largest measured destabilization was 1.1 kcal mol-1 for a Val to Ile change. Two or three such mutations could thus account for the whole decrease in stability in ZSPA-1 compared to the Z domain. Another explanation of the dynamic behaviour of ZSPA-1 has been presented by Favrin and colleagues (Favrin et al. 2004). Off-lattice folding simulations of the Z domain and ZSPA-1 were able to reproduce the lower stability, less cooperative melting transition and reduced ∆Cp° for unfolding of ZSPA-1. The data also indicated a reduced helix propensity for helix 1, probably due to the glycine residue in position 13 in ZSPA-1. This residue would thus provide an interesting target for further mutational studies.

10 Investigation of the thermodynamics of the Z:ZSPA-1 complex formation (IV) Due to the molten globule behaviour of the ZSPA-1 affibody, a thermodynamic investigation of this system was a challenging task. The notion of a non-linear relationship between the measured ∆H°bind and temperature indicated a process with coupled equilibria. As a first attempt to resolve these processes, the thermodynamics of the Z domain and the ZSPA-1 unfolding was investigated using van’t Hoff analysis. The free energy of unfolding for the Z domain agrees reasonable well with other published data (Cedergren et al. 1993; Bai et al. 1997; Myers and Oas 2001). The unfolding of the ZSPA-1 affibody is accompanied with a reduction of ∆G°unfold of approximately 3 kcal mol-1 compared

Christofer Lendel

51

to the Z domain. In addition, there is a lower ∆Cp°unfold and a higher ∆S°unfold for the affibody, as discussed in the previous chapter. Provided the thermodynamic data for ZSPA-1 unfolding, this process was successfully separated from the measured binding thermodynamics using the following equations: ∆H°bind = ∆HITC – f unfold ∆H°fold

(6)

∆G°bind = – RT ln ((1+Kunfold)/Kdapp)

(7)

where the subscript ‘ITC’ and the superscript ‘app’ denote the measured values and funfold is the fraction unfolded protein. The corrected binding data shows a linear relation between ∆H°bind and temperature, indicating that the unfolding was the main source of non-linearity (figure 21). This is further strengthened by the results in presence of TMAO or betaine, for which only small or no corrections are required to obtain linearity.

Kunfold

5

Kd

DH (kcal/mol)

0 DHbind

-5 -10 -15 10

DHfold

15

20

25

30

35

Temperature (ºC) Figure 21. Illustration of the coupled folding and binding mechanism together with the observed enthalpies for the Z:ZSPA-1 complex formation at different temperatures (open diamonds) and the enthalpies after correction for ZSPA-1 folding (solid circles). Data measured at pH 5.7. See also figure 3 in (IV). Figure kindly provided by T. Härd.

The unfolding-corrected thermodynamic data contains contributions from both the binding and the stabilization of the molten globule, which should rather be considered as a part of the association mechanism than a coupled process. The complex formation is characterized by favourable contributions from both enthalpy and entropy and a large negative change in heat

52


capacity, indicating the importance of desolvation effects. This is in agreement with the hydrophobic interface observed in the structure of the complex. In comparison with the compilation of thermodynamic data for protein-protein interactions by Stites (Stites 1997), these results seem normal. Many interactions are favoured by both enthalpy and entropy. A negative heat capacity change in the region 100 - 500 cal mol-1 K-1 is also a common feature. However, this statistics shows a large diversity, implying that almost anything is possible. The complex structure did not provide any clues about why the affinity was not as high as in other affibody complexes (Gunneriusson, et al. 1999a; Nord et al. 2001). There is also a 100fold affinity difference compared to the Z:Fc complex (Jendeberg et al. 1995), even though the interface is very similar to the Z:ZSPA-1 complex. The correction for the unfolding equilibrium only resulted in small changes in the binding free energy, so this cannot be the only reason for the moderate affinity. In general, coupled folding reactions will only have small effects on the free energy of binding close to or below Tm (Cliff et al. 2005). The entalpic and entropic contributions can, however, still be large. Structure based calculations of a hypothetical complex formation, where the free affibody adopts exactly the same structure as in the complex, resulted in values in reasonable agreement with the experimental data for ∆H°bind and ∆Cp°bind but not for ∆S°bind. The calculated –T∆S°bind values were 17-27 kcal mol-1 more favourable than the experimental data, indicating that the conformational stabilization of the molten globule upon binding reduces the binding affinity. In order to support this suggestion the change in conformational entropy for stabilizing the molten globule state was also estimated using two other methods. The first approach was based on the change in helicity for ZSPA-1 upon complex formation measured by CD. The change in CD signal is consistent with a coil-to-helix transition of 12 residues. The exact nature of these residues is not known since the reduced helicity originates from transient unfolding of helices 1 and 2 (III). However, using the coil-to-helix transition data from D’Aquino and colleagues (D’Aquino et al. 1996) for 12 residues with the average composition of the residues in helices 1 and 2, a conformational entropy penalty of 16 kcal mol-1 was obtained. The second approach used the assumption that the desolvation entropy is zero at 112 °C, as described in chapter 3. Using the experimental data for ∆Cp°bind and ∆S°bind the contribution from conformational entropy was calculated from:

Christofer Lendel

–∆Sconf = ∆Cp,bind ln(T/385) + ∆Srt - ∆S°bind(T)

53

(8)

with the rotational and translational entropy (∆Srt) set to –8 cal mol-1 K-1 as described in (IV). The resulting value of –T∆S°conf = 23 ± 2 kcal mol-1 is in reasonable agreement with the value obtained from the first approach and in the same range as the overestimation of the entropic effect from the structure based calculations. Hence, the reason for the moderate affinity of the Z:ZSPA-1 complex is an entropic penalty associated with the stabilization of the molten globule state. The importance of the conformational stability of the free protein for the affinity was evident also in the construction of a minimized Z domain done by Wells and colleagues (Braisted and Wells 1996; Starovasnik et al. 1997). When constructing their two-helix variant they observed a strong correlation between helical content and binding affinity. Stabilization of the construct with a disulfide linkage improved the affinity further. This was mainly due to increased on-rate, which is consistent with increased stability of the free state. Furthermore, several thermodynamic investigations of protein-peptide interactions have shown that these systems often experience unfavourable change in conformational entropy upon association (Welfle et al. 2001; Welfle et al. 2003; Cliff et al. 2005).

11 Investigation of the ZTaq:anti-ZTaq complex (V, VI) With the extensive data available for the Z:ZSPA-1 system an interesting question was which similarities and differences would be found in another affibody-target complex. In addition to ZSPA-1, two other anti-idiotypic affibodies, anti-ZTaq and anti-ZIgA, were isolated by Eklund and colleagues (Eklund et al. 2002). These were selected using the ZTaq (Gunneriusson et al. 1999a) and ZIgA (Rönnmark et al. 2002) affibodies as panning targets. Since the dissociation constants were similar for these complexes, the choice to work with the ZTaq:anti-ZTaq system was based on the two tryptophans present in the ZTaq affibody. Neither ZIgA nor anti-ZIgA have any tryptophan or tyrosine residues, which seriously complicates measurements of protein concentration.

54


Taq DNA polymerase-binding affibodies with micromolar affinities were selected already in the first tests of the affibody methodology (Nord et al. 1997). One of these was subjected to affinity maturation of helix 2 to improve the binding strength (Gunneriusson et al. 1999a). The best binder (denoted ZTaq S1-1 by Gunneriusson et al.) from this library of second-generation affibodies showed a Kd of 25 nM. The unaltered helix 1 together with the conservation of certain positions indicate that the matured variant recognizes the same binding site on Taq DNA polymeras as the original construct (Gunneriusson et al. 1999a). This affibody was subsequently applied as panning target to obtain an anti-ZTaq affibody (Eklund et al. 2002). The selection was performed using standard low pH phage elution protocol without any competition. Five binders were isolated and surface plasmon resonance binding studies were used to identify the best variant. Anti-ZTaq (i.e. the variant denoted anti-ZTaq S1-1 clone 16:4 by Eklund et al.) binds the target ZTaq with a Kd of 0.1 µM (J. Dogan and C. Lendel, manuscript in preparation).

Fraction unfolded

1.0

0.5

0

0

20

40

60

80

100

O

Temperature ( C) Figure 22. Thermal denaturation of the Z domain (solid squares), ZSPA-1 (solid circles), ZTaq (triangles) and anti-ZTaq (solid diamonds) monitored by CD at 222 nm.

Preliminary biophysical characterization of the free proteins did not show the molten globular behaviour to the same extent as seen for ZSPA-1. The 15N-HSQC spectra are consistent with fully structured proteins, the melting temperatures are around 55 °C (figure 22), and only minor ANS fluorescence enhancement is observed. There are, however, indications of dynamic events in both proteins. In anti-ZTaq, the exchange of amide hydrogen atoms is fast (all amide resonance disappear from the

15N-HSQC

in less than 15 min) and in ZTaq, R2

Christofer Lendel

55

relaxation measurements indicate exchange broadening for a few residues in helix 1. Surprisingly, the helix content of ZTaq, judged from the CD spectrum, seem to be similar to ZSPA-1 (mean residue ellipticity = -17 200 deg cm2 dmol-1 at 222 nm and 25 °C) while the helicity of anti-ZTaq is at the level of Z domain (mean residue ellipticity = –28 400 deg cm2 dmol-1 at 222 nm and 25 °C). Since these values do not correlate with the fraction helix seen in the determined structures (vide infra), the CD signal must be affected by dynamic events. In addition, it should be noted that the CD measurements are sensitive to the protein concentration. Typical errors of 5-10 % in concentration measurements result in the same error for the CD signal amplitude. Both ZTaq and anti-ZTaq have more polar to non-polar substitutions than ZSPA-1. The fractions exposed hydrophobic surface are, however, slightly smaller, 62 % and 60 % for ZTaq and antiZTaq respectively compared to 64 % for ZSPA-1. Secondary structure prediction algorithms (Combet et al. 2000) do, in fact, indicate decreased propensity for formation of helix 1 in ZTaq (figure 23), which is consistent with the lower CD signal and the observed dynamics. Anti-ZTaq has almost perfect helix propensity but is not as stable as the Z domain. Hence, helix propensity is important but not the only determinant of stability. Moreover, the increased stability of these proteins compared to ZSPA-1 are important results since they imply that randomisation of the Z domain not necessarily results in molten globule proteins.

Z domain ZSPA-1 ZTaq anti-ZTaq

10 20 30 40 50 | | | | | cccccchhhhhhhhhhe?ccccchhhhhhhhhhhccccchhhhhhhhhhhhhhhcccc ccccc?hhhhhhcceeee?ccccccchhheeeeeccccchhhhhhhhhhhhhhhcccc cccccchhh?hhheeeecccccc?hhhhhhhhhhcccc?hhhhhhhhhhhhhhhcccc cccccchhhhhhhhhhhcccccchhhhhhhhhhhccccchhhhhhhhhhhhhhhcccc

Figure 23. Secondary structure prediction for the Z domain and the three studied affibodies using the secondary consensus prediction method (Combet et al. 2000); h, alpha helix; e, extended strand; c, random coil; ?, ambiguous state.

The quality of the NMR data for the free proteins allowed for determination of their structures. Such complete structural basis, involving the structures of both the complex and the free subunits determined at identical experimental conditions, is highly interesting, not only for understanding these proteins, but also for testing docking algorithms and for the understanding of protein-protein interactions in general. The experimental conditions were this time chosen to be more similar to those at which the phage-display selection had been

56


performed. The temperature was thus 25 °C, pH 6.4 and 50 mM NaCl was added to the phosphate buffer. The approach for assigning the NMR resonances was the same as employed for the Z:ZSPA-1 complex and essentially complete assignments were obtained for 1H, 13C and 15N

atoms for both proteins in free and complex-bound states (V).

The structure of the complex was determined from in total 2685 NMR-derived restraints, corresponding to 23.1 restraints per residue (VI). The r.m.s.d. for all heavy atoms in the ordered parts is 0.61 ± 0.06 Å (residues 4-55 in ZTaq and 6-55 in anti-ZTaq). Interestingly, the two proteins have found each other’s randomised surfaces even though no competitive elution was employed during the selection. However, self-recognition leading to aggregation could still provide a bias toward this region, as discussed in chapter 8. The relative orientation of the subunits is almost perpendicular and the interface buries a total surface area of 1672 Å2, with a very high fraction (70 %) being non-polar. This is, in fact, similar to what is seen in cores of monomeric proteins (Janin et al. 1988). Other relevant structural data includes 13 hydrogen bonds and 174 interface heavy atoms, belonging to 46 residues (figure 24). Anti-ZTaq uses all randomised positions for inter-molecular contacts and is thus optimised in that aspect. Some fascinating features are found in the interface: i) The side chain of Arg35 (ZTaq) is coordinated by up to four hydrogen bonds to backbone carbonyls of anti-ZTaq, which are confirmed by two NηH2 resonances of the arginine being visible in the 15N-HSQC. These are among the most rare resonances to be observed in proteins according to the BioMagResBank (http://www.bmrb.wisc.edu/). ii) The glycines Gly10 (ZTaq) and Gly14 (antiZTaq) come very close to each other in the interface. This increases the number of contacts in the surrounding and offers possibilities for hydrogen binding using the Hα atoms of the glycine residues. Although the energetic importance of such hydrogen bonds is a matter of discussion, glycines seem to be especially suitable for donating Hα hydrogen bonds due to the higher CαHα charge separation and the ability to get closer to the acceptor than any other amino acid residue (Senes et al. 2001). iii) A threonine residue (Thr13 in ZTaq) is found in the middle of the hydrophobic core of the interface. This is surprising since burying hydroxyl groups in hydrophobic environments is associated with an energetic penalty. The reason for the selection of a threonine in this position might be that ZTaq was selected to bind Taq DNA polymerase, and not anti-ZTaq, or it could be a consequence of the limited library size. This residue provides a very interesting target for a mutational study.

Christofer Lendel

57

a V27 K28 N18

F17

I31

D32

W14 L34

T13 W11

R35

G10

L9 N6

K7 F5

N3

b

S24 L25

L22

V28 L19

V27

I16

N32

M17 R18

I31

E15 G14

L34

I13

R35

R9 I11

V10

N6 K7

Figure 24. The ZTaq:anti-ZTaq interface. (a) Helices 1 and 2 of ZTaq upon a surface representation of antiZTaq. The interacting side chains are shown. (b) Helices 1 and 2 of anti-ZTaq upon a surface representation of ZTaq. Residues involved in backbone or side chain contacts are denoted.

58


The most obvious difference between the Z:ZSPA-1 and ZTaq:anti-ZTaq complexes is the relative orientation. It is fascinating that these proteins, still sharing at least 78 % of the sequence, employ two completely different binding modes. According to Chothia et al. (Chothia et al. 1981), helices pack against each other with approximately 20°, 50° or 105° relative orientation. Most common in the analysed data set of protein structures was the 50° variant and it was noticed that alternative packing is often associated with the presence of a small amino acid, such as alanine or glycine. Both proteins in the ZTaq:anti-ZTaq complex present glycine residues in the interface, which might be the reason for the observed helix orientation. Beside the packing issue, the structural characteristics in terms of surface area, hydrophobicity, hydrogen bonds etc., are similar (Table 1). The influence of the small differences on the affinity is not obvious. For instance, one or two hydrogen bonds could, in theory, account for the whole difference in binding energy. Table 1. Structural data for protein-protein complexes. Z :ZSPA-1

ZTaq:anti-ZTaq

B:Fc

Statistics for 75 complexes a

Total interface area (Å2)

1600 ± 400 b

1632

1672

1234 d

Non-polar area (%)

64

70

59 a

56 ± 6

Hydrogen bonds

10

13

2

a

10.1 ± 4.8

Interface atoms

186

174

146

a

174 b,c

32

46

Interface residues

20 d

44 b,c

aData

from Lo Conte et al. 1999. with ‘standard size’ interface cNo standard deviation given in reference. dData from Deisenhofer 1981. bComplexes

The structures of the free proteins were determined from 1162 and 1315 NMR-derived restraints, corresponding to 20.2 and 22.7 restraints per residue for ZTaq and anti-ZTaq, respectively (VI). The r.m.s.d.s of all heavy atoms in the ordered parts are: 0.74 ± 0.09 Å for residues 6-55 in ZTaq and 0.66 ± 0.05 Å for residues 5-55 in anti-ZTaq. Both proteins adopt three-helix bundle folds. There are, however, small deviations in the orientation of helix 1 compared to the complexed structures and to the originating Z domain. The angles between helices 1 and 2 seem to be slightly smaller in the free subunits, which is interesting since the orientation of helix 1 in protein A domains has been debated (as described in chapter 6.4).

Christofer Lendel

59

The reorientation of the first helices upon complex formation makes the interaction surfaces slightly larger. The changes in accessible surface areas of the interface regions is in fact larger than the total changes for the proteins. The availability of all the structural components also reveals events of extensive induced fit on the side chain level. The aromatic residues Trp11, Trp14 and Phe17 in ZTaq move up to 9 Å to fit into the binding surface of anti-ZTaq (figure 25a) and the methyl group of Met17 in anti-ZTaq is turned around almost 180° to point into a cavity in the ZTaq interface (figure 25b). In addition several other side chains experience small or medium rearrangements and/or transitions into more defined conformations. These data clearly state that models describing protein-protein interactions will not be accurate unless some level of plasticity is implemented. Z

a

b

W11 Z

F17

Z

W14Z

A

M17

Figure 25. Examples of induced fit upon ZTaq:anti-ZTaq complex formation. (a) Trp11, Trp14 and Phe17 in ZTaq rearrange to fit into the anti-ZTaq binding surface. Green color denotes positions in the free state and blue in the complex. (b) The methyl group of Met17 in anti-ZTaq rotates almost 180° when binding to ZTaq. The yellow side chain is the orientation in the free protein and the red the position in the complex.

The structural data of two different affibody complexes might reveal some general aspects of the binding mechanisms in these proteins. A common feature of the two complexes, which is also shared with the B:Fc complex, is the hydrophobic nature of the interaction. The important residue Ile31 in SPA domains (Jendeberg et al. 1995) has a consequent central position and becomes completely buried in all four proteins. Another SPA hot spot residue, Phe13 (DeLano et al. 2000), is mutated in the affibody library and even though there is no real hydrophobic conservation, highly polar residues seem to be depleted from this position in isolated target binding affibodies (see figure 10). As pointed out in (VI), no real conclusions can be made

60


from this small set of structures. Additional mutational data, preferably from several affibodies, is needed to confirm these observations. The similarities between the interfaces of the Z:ZSPA-1 and B:Fc complexes raise the question if the ZTaq:Taq DNA polymerase interaction could be modeled from the ZTaq:anti-ZTaq complex and the available crystal structure of Taq DNA polymerase (Murali et al. 1998). As observed for the Z:ZSPA-1 and B:Fc cases, the interfaces have no similarity on secondary or tertiary structure level. Rather the relative orientation of some key residues and the surface topology should be considered. However, the surface plasticity discussed above makes it unlikely that the interaction surface in free Taq DNA polymerase would show any similarities with the bound variant and since the hydrophobicity seems to be the most pronounced feature of the interface, it is not possible to search for specific key residues either. Many combinations of hydrophobic residues could do. The prediction of ZTaq:Taq DNA polymerase binding is thus not straightforward. However, the ZTaq:anti-ZTaq data might be useful for scoring solutions obtained from computational docking algorithms.

b

Time (min) 0

33

67

100

133

0.0

0.0

DH (kcal/mol)

-0.5 -1.0 0

kcal / mole of anti-ZTaq

(mcal / sec)

a

-5 -10 0.0

0.5

1.0

1.5

[anti-ZTaq] / [ZTaq]

2.0

2.5

4.0 8.0 -12.0 -16.0 10

15

20

25

O

30

35

Temperature ( C)

Figure 26. (a) A representative ITC measurement of the ZTaq:anti-ZTaq complex formation at 25.4 °C. 225 µM anti-ZTaq was titrated into a solution containing 23.1 µM ZTaq. The experiments were performed as described in (III and IV) with the same buffer conditions as described in (V and VI). (b) Measured ∆Hº plotted against the temperature. R2 for the linear fit is 0.99.

A thermodynamic investigation of the complex formation (J. Dogan and C. Lendel, manuscript in preparation) indicates a highly exothermic reaction and a large negative change in heat capacity (larger than for Z:ZSPA-1) (figure 26). This is in agreement with the extra hydrogen bonds and the more hydrophobic character of the interface. The total binding

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entropy is small and the reason seems to be a cancellation between favourable desolvation entropy and unfavourable conformational entropy. Hence, the dynamic events observed in the free proteins might result in an entropic penalty in a similar way as for the Z:ZSPA-1 complex formation.

12 Concluding remarks 12.1 Affibody-target recognition First, the presented work has confirmed the supposed binding mode of the affibodies. They use the randomised surface and the varied positions provide affinity and specificity to the complex. The investigations strongly indicate that the hydrophobic effect is the main driving force for target affinity in the studied affibodies, while a wide range of electrostatic interactions of different nature provide additional binding energy and regulate the target specificity. As DeLano and co-workers have noted (DeLano et al. 2000), there are intrinsic binding properties of the Fc surface that binds protein A. It is not unlikely that corresponding properties are found in the protein A domains, especially since hot spots often bind to other hot spots (Li et al. 2004). The properties characterizing these intrinsic binding surfaces are accessibility, hydrophobicity and adaptability (DeLano et al. 2000). It is obvious from the presented results that the binding surfaces of the affibodies exhibit all these properties. Hence, it seems like the affibodies have inherited certain intrinsic binding properties from the ancestral protein A. Changing 13 residues in such a small protein as the Z domain could certainly affect the stability. We have demonstrated that the presence of an unfolding equilibrium due to reduced stability of the native(-like) state has only minor effects of the binding free energy. Increased flexibility of the free affibody molecules, on the other hand, can seriously decrease the binding affinity due to loss in conformational entropy upon target binding. It would thus be very interesting to investigate if low conformational flexibility is the reason for the high affinity (Kd in nM, or even pM region, P.-Å. Nygren, personal communication) observed for certain affibodies.

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12.2 Protein stability Even though the forces and their relative importance for protein stability are quite well established, the present level of understanding can still not provide a detailed description of the energetics. Especially the roles of amino acids in the protein-water interface seem to be difficult to elucidate. Considering all the changes made in ZSPA-1 there are a number of things that will affect the stability, including introduction of additional positive charges, reduction of the number of polar (non-charged) residues, introduction of a glycine residue, increased surface hydrophobicity and changed helix propensity. Extensive experimental data is required to completely separate these effects. As obvious from the literature, molten globules can show large variations in structural content, timescales of dynamics and similarity to the native structure. However, the common property for molten globule proteins is the conformational degeneracy of the native state. As demonstrated, NMR offers many possibilities to characterize the structure and interconversion dynamics. Studies like this, where these states are investigated on a molecular level will continue to provide important insights in the mechanisms of folding and misfolding of proteins.

12.3 Protein-protein interactions Protein-protein interactions could, compared to for instance folding, seem less complex. However, the presented data shows that even binding events between small and relatively simple proteins can show high complexity involving many specific and fascinating interactions, intricate events of induced fit and coupled folding reactions. We have clearly demonstrated the importance of careful investigations of all participating components. Neither the absolute affinities nor the relative binding strengths of the two investigated complexes can be understood from solely the complex structures or the structural properties of the free subunits, but taken together, at least a ranking of the relative affinities is possible. However, to correlate the structural data with the absolute affinities the investigations must also be accompanied by thermodynamic measurements. For a better understanding of the mechanisms of proteinprotein interactions more systems must be studied in this complete manner. The correlations between interfacial architecture, conformational changes and thermodynamic properties might then become clear.

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13 Acknowledgements I would like to thank: Torleif for being my supervisor (even though I did not follow you to Göteborg), for your excellent scientific guidance and for all the help with this thesis. Per-Åke for accepting to be my supervisor when Torleif moved, for giving me many new perspectives of my research and for valuable comments on this thesis. Elisabet, Jakob, Vildan and Alex for productive collaboration and invaluable contributions to this work. Peter for your contributions to the Z:ZSPA-1 project, your NMR knowledge that you kindly share, for being an excellent supervisor during my ‘X-jobb’ and for your FGB-application. Magnus H, Helena, Anders H and Malin that have also considerably contributed to the success of the affibody project. Helena is also acknowledged for keeping the group going when Torleif moved. All members of the former structural biochemistry group that have not been mentioned above: Alexandra, Anders Ö, Anja, Esmeralda, Henrik, Inger, John, Lotta, Magnus W-W, Martin, Niklas, Per-Åke and Susanne. The BioCat group for letting me join you during the last months. All new colleagues at floor 2 and all old colleagues at NOVUM. FGB for financial support. I will also take the opportunity to mention a few people that more or less are responsible for the road leading to this thesis: Morbror Anders, the first (non-medical) doctor I knew. Even though it is now almost 10 years since you left us, you have been a great inspiration during this work. My science teacher in ‘högstadiet’, Anders Erixon, that encouraged wild experiments and fed my curiosity. My chemistry teacher in ‘gymnasiet’, Ylva Pettersson that taught me how to ‘understand’ organic chemistry as well as biochemistry. István Furó that introduced me to the fascinating world of NMR. I am also very grateful to: My mother, father, brother and sister, for always supporting me and helping me with the dissertation party. Göta & Staś for all your help during the last months. Stina for all your love and patience. Without you this thesis would not have been finished in time. I love you more than ever! Alva for the inspiration during the writing of this thesis and for making my dissertation the second most important event this year.

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