TIGRAN V. CHALIKIAN AND KENNETH J. BRESLAUER*. Department of Chemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08855-1)939.
Proc. Natil. Acad. Sci. USA Vol. 93, pp. 1012-1014, February 1996 Biochemistry
Compressibility as a means to detect and characterize globular protein states TIGRAN V. CHALIKIAN AND KENNETH J. BRESLAUER* Department of Chemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08855-1)939
Comnninicated by SteplVen J. Benkovic, Pennsylvania State University, University Park, PA, October 23, 1995
ABSTRACT We report compressibility data on singledomain, globular proteins which suggest a general relationship between protein conformational transitions and Ak', the change in the partial specific adiabatic compressibility which accompanies the transition. Specifically, we find transitions between native and compact intermediate states to be accompanied by small increases in ks of + (1-4) x 10-6 cm3 g-'bar-l (1 bar = 100 kPa). By contrast, transitions between native and partially unfolded states are accompanied by small decreases in ks of -(3-7) x 10-6 cm3g- 'bar-', while native-to-fully unfolded transitions result in large decreases in k' of -(18-20) x 10-6 cm3.g-'-bar-'. Thus, for the single-domain, globular proteins studied here, changes in ks correlate with the type of transition being monitored, independent of the specific protein. Consequently, k' measurements may provide a convenient approach for detecting the existence of and for defining the nature of protein transitions, while also characterizing the hydration properties of individual protein states.
inaccessible core and its coefficient of adiabatic compressibility, OM (7): kM
=
13MVM/M,
[2]
where M is the molecular weight of the protein. The hydration contribution, kh, reflects the decrease in the compressibility of the solvent which results from interactions between the surface atomic groups of the protein and the surrounding water molecules and can be calculated from the expression (7, 8):
kh =
m-
SAj&j,
[31
The intrinsic contribution, kM, reflects the imperfect packing of the polypeptide chain(s) within the solventinaccessible interior of proteins. As shown in Eq. 2 below, the value of kM is related to the volume, VM, of the water-
where SA, is the solvent-accessible surface area of the ith residue and Ksi is the compressibility contribution of 1 A2 of this surface of the ith residue. In general, for globular proteins, the sum -i SA,Ksi yields negative values for the hydration contribution, kh, to the partial compressibility, although the sign and magnitude of Ksi for an individual residue depend on the experimental conditions and the type of the residue (8). Based on these definitions and the relationship expressed by Eq. 1, two generalizations emerge. First, the more rigid the interior of a globular protein (in other words, the tighter its internal packing), the smaller its intrinsic compressibility, kM, and, consequently, the smaller the total partial specific adiabatic compressibility, k°, of the protein. Second, the more protein atomic groups that are exposed to the solvent (in other words, the higher the total solvent-accessible surface area), the more negative the hydration contribution, kh, and, consequently, the smaller the total value of k'. Given these interrelationships, we present below the results of ks measurements we have conducted on a range of globular proteins, beginning with cytochrome c. For cytochrome c, we find the partial specific adiabatic compressibility, k', of the protein to vary significantly with the nature of the protein conformational state present in solution (9). The following rank order emerges: k' (CI) > ks (N) > k' (PU) > k' (FU). To be specific, for cytochrome c the acidinduced N-to-Cl transition is accompanied by a small positive change in ks of + 1.7 x 10-6 cm3g-'-bar-', and the acidinduced N-to-PU transition is accompanied by a small negative change in k' of -3.9 x 10-6 cm3g- '-bar-l. We calculate the N-to-FU transition to be accompanied by a large negative change in k' of -20 x 10-6 cm3 g-l bar-1. These Ak' values can be rationalized in terms of changes in a positively contributing intrinsic component, kM, and a negatively contributing hydration component, kh, with the latter diminishing with increasing protein hydration (ref. 8 and references therein; ref. 9). For cytochrome c, we find the following trend in kh as a function of protein state: kh(N) > kh(CI) > kh(PU) > kh(FU). The positive contribution of kM increases as the interior packing of a protein decreases. For cytochrome c, we find that
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Abbreviations: N, native; CI, compact intermediate; PU, partially unfolded; FU, fully unfolded; MG, molten globule. *To whom reprint requests should be addressed.
Depending on solution conditions, globular proteins can assume a range of conformational states, including native (N), compact intermediate (CI), partially unfolded (PU), and fully unfolded (FU) (1-5). The N state of a globular protein is characterized by a unique tertiary structure, with tightly packed amino acid residues buried within a solventinaccessible protein interior. By contrast, the CI state, which includes molten globules (MGs), is characterized by a lack of rigid tertiary structure, a high content of secondary structure, and a sizable core of nonpolar groups which are packed less tightly than in the N state (4, 5). The PU state, which we define as the ensemble of partially unfolded conformations that can be detected between the CI and FU states, is characterized by a lack of both tertiary and secondary structural elements, with a highly fluctuating and very loosely packed hydrophobic core. The FU state is random coil-like, with all atomic groups being solvent accessible (2). Clearly, it would be very useful to identify an experimental observable that could both detect and uniquely characterize protein states. Based on the results reported here, we believe that the partial specific adiabatic compressibility, k', of a protein fulfills these requirements. Recall that the k' of a solute is a linear function of the isothermal pressure derivative of its partial specific volume, v° (6). For globular proteins, the value of k' is the sum of a positive intrinsic contribution, kM, and a negative hydration contribution, kh (7, 8):
ko kM + kh-
[1]
1012
Proc. Natl. Acad. Sci. USA 93 (1996)
Biochemistry: Chalikian and Breslauer kM increases for the N-to-Cl and N-to-PU transitions but is reduced almost to zero for the N-to-FU transition (9). In short, our data on cytochrome c reveal that the value of k' and its components, kM and kh, depend on the predominant protein state present in solution. To assess the generality, if any, of a relationship between the sign and magnitude of k' and the conformational state of a protein, we also have measured changes in compressibility that accompany conformational transitions in three other small, single-domain, globular protein systems. The resulting data, including those for cytochrome c, are listed in Table 1, along with the few k' values that can be found in the literature. The following features should be noted. All four of the N-to-Cl (e.g., MG) transitions are accompanied by small increases in ko of +(1-4) x 10-6 cm3g-'-bar-'. All three of the N-to-PU transitions are accompanied by small decreases in ko of - (3-7) The N-to-FU transition of ribonuclease x 10-6 cm3 g- I barA is accompanied by a large decrease in k' of -18 x 10-6 cm3. g- lbar- 1, which is in excellent agreement with our calculated value of -20 x 10-6 cm3 g-l bar- for the N-to-FU transition of cytochrome c (9). In the aggregate, these results suggest a general relationship between the sign and magnitude of Ako and the protein state. As discussed below, the origins of such a general relationship can be understood in terms of the properties of the various states that globular proteins can assume. The N state of a globular protein is the most compact conformation amongst the possible thermodynamic states (N, CI, PU, and FU). This feature causes the N state to exhibit the lowest solvent-accessible surface area, SA, and, consequently, the highest (least negative) hydration contribution, kh, to the partial compressibility. The native state also exhibits the largest volume for the water-inaccessible core, VM, as well as the most tightly packed core, with the coefficient of adiabatic compressibility, g3M, of this core being low and close to that of organic solids (7). When this N state of a globular protein is converted into a CI state such as a MG (the N-to-CT transition), some of the
buried residues of the N state become exposed to the solvent in the CI state (4). Consequently, the N-to-Cl transition should be accompanied by an increase in the accessible surface area, SA, thereby resulting in a decrease in the hydration contribution, kh. Consistent with this expectation, we find the acid-induced N-to-MG transition of cytochrome c to be accompanied by an -2-fold decrease in kh (9)Furthermore, although the volume of the water-inaccessible core, VM, also should decrease upon a N-to-Cl transition, the intrinsic coefficient of adiabatic compressibility, ,BM, of the core should increase due to loosening of the interior packing. Consistent with this expectation, we find the acid-induced N-to-MG transition of cytochrome c to be accompanied by a 4.5-fold increase in ,BM (9). As we have shown for cytochrome c (9), this increase in ,BM prevails over the decrease in VM, thereby resulting in a net increase in the intrinsic contribution, kM, to the partial compressibility (see Eq. 2). For the N-to-MG transitions examined here, this increase in kM is higher in absolute value than the decrease in kh. Consequently, consistent with Eq. 1, we observe a net increase in k' of (1-4) x 10-6 cm3 g- Ibar- i for the N-to-CI transitions of the globular proteins we have examined (see Table 1). When the N state of a globular protein is converted into a PU state (the N-to-PU transition), a substantial increase in SA should occur, with about 70% of the surface area expected for a fully extended chain becoming exposed to the solvent (9, 19). This exposure should cause a large decrease in kh, although a water-inaccessible core, probably mostly hydrophobic, still is preserved. The intrinsic volume, VM, of this preserved core in the PU state, however, should be small, while the coefficient of adiabatic compressibility, 13M, of the core should be high due to it being loosely packed. Consequently, although the total value of the intrinsic contribution, kM, may increase upon the N-to-PU transition [as we find for the acid induced N-to-PU transition of cytochrome c (9)], the absolute value of the decrease in kh due to solvent exposure prevails over any increase in kM, since we observe moderate net decreases in ks
Table 1. Partial specific adiabatic compressibilities, k', of globular proteins and the changes in compressibility, which accompany conformational transitions at 25°C Value x 106, cm3-g- l barko Aks Transition 1.7 2.5 N-to-MG of cytochrome Ca; acid-induced at 25°C in 200 mM CsCl 2.7 1.1 N-to-MG of a-lactalbumin6; acid-induced at 25°C in 2 mM CaC12 3.2 3.9t N-to-Cl of a-chymotrypsinogen Ac; temperature-induced at pH 2.0 in 10 mM NaCl
2.6t N-to-Cl of ribonuclease N-to-PU of cytochrome N-to-PU of cytochrome N-to-PU of myoglobing;
Ad; temperature induced
at pH 1.9
Cc; acid-induced at 250C Cf; base-induced at 25°C acid-induced at 25°C in 10 mM KCl
0.9 2.5 2.5 7.1
4.Ot -3.9 -3.8 -6.8 -4.8 -18
Aks,
Ref. 9 * * 10 11 9 22 12 13 11
0.9 N-to-FU of ribonuclease Ah; Gdn HCl-induced at 25°C in 0.2 M CsCl aCharacterized as a N-to-MG transition by Ohgushi and Wada (14). bCharacterized as a N-to-MG transition by Dolgikh et al. (15). cCharacterized as a N-to-CT transition based on CD measurements (*) at acidic pH, which reveal that heat-denatured a-chymotrypsinogen A lacks tertiary structure while retaining secondary structure. '-Characterized as a N-to-CT transition based on CD spectra, small-angle x-ray scattering, and Fourier transform infrared spectroscopy (16, 17), which reveal that thermally denatured ribonuclease A has compact dimensions and retains residual secondary structure. cCharacterized as a N-to-PU transition based on CD measurements (9, 18), which reveal that, at low salt concentrations, the acid-denatured state of cytochrome c lacks both secondary and tertiary structure. tCharacterized as a N-to-PU transition based on CD measurements (22), which reveal that, at low salt concentrations, the base-denatured state of cytochrome c lacks both secondary and tertiary structure. gCharacterized as a N-to-PU transition based on CD measurements (*), which reveal that, at low salt concentrations, the acid-denatured state of myoglobin lacks both secondary and tertiary structure. hCharacterized as a N-to-FU transition based on the coincidence between the reported (12) and calculated (9) values of Aks. *T.V.C., V. S. Gindikin, D. Anafi, and K.J.B., unpublished data.
tExtrapolated
to 25°C.
1013
1014
Biochemistry: Chalikian and Breslauer
of (3-7) x 10-6 cm3.g-lbar- for the N-to-PU transitions listed in Table 1 (see Eq. 1). When the N state of a globular protein is converted into a FU state (the N-to-FU transition), complete solvent exposure of the formally buried residues occurs. In the absence of a solvent-inaccessible core, k' of the FU state should be determined exclusively by a large negative kh term. Consistent with this expectation, we find the N-to-FU transition to be accompanied by a large decrease in k' of about 20 x 10 -6 cm3.g- 'bar- l As noted above, our results suggest a general relationship between the sign and magnitude of Ak' and the protein state. We propose that this relationship can be understood in terms of the foregoing discussion. Significantly, however, the existence of the empirical relationship and its utility do not depend on the details of our interpretation. In summary, we find that changes in k' for single-domain, globular proteins appear to correlate with the type of transition being monitored, independent of the specific globular protein under study. This property of Ak' may prove to be a common thermodynamic feature of small globular proteins, similar to the convergence of the changes in enthalpy (at 110°C) and entropy (at 112°C) which accompany heat denaturation of such proteins (19-21). Consequently, ks measurements may provide a convenient approach for detecting the existence of and for defining the nature of protein transitions, while also characterizing the hydration properties of individual protein states (8). 1. Tanford, C. (1968) Adv. Protein Chem. 23, 121-282. 2. Tanford, C. (1970) Adv. Protein Chem. 24, 1-95. 3. Dill, K. A. & Shortle, D. (1991) Annu. Rev. Biochem. 60, 795825. 4. Ptitsyn, 0. B. (1992) in Protein Folding, ed. Creighton, T. E. (Freeman, New York), pp. 243-300.
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