Finding the right balance a personal journey ... - Wiley Online Library

THE FEBS ⁄ EMBO WOMEN IN SCIENCE LECTURE

Finding the right balance – a personal journey from individual proteins to membrane-embedded motors Based on a lecture delivered at the 36th FEBS Congress in Torino, Italy, June 2011 Carol V. Robinson Department of Chemistry, University of Oxford, UK

Keywords gas phase micelles; gas phase structural biology; HSP90 reaction cycle; mass spectrometry; membrane-embedded motors; protein complexes; V-type ATPases Correspondence C. V. Robinson, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK Fax: +44 1865 275410 Tel: +44 1865 275410 E-mail: [email protected] (Received 28 October 2011, revised 7 December 2011, accepted 12 December 2011) doi:10.1111/j.1742-4658.2011.08460.x

It is now more than 20 years since the prophetic words of John Fenn, announcing his discovery, stated that ‘Electrospray spectra have been obtained for biopolymers including oligonucleotides and proteins, the latter having molecular weights up to 130 000, with as yet no evidence of an upper limit’ (Fenn JB, Mann M, Kai Meng C, Fu Wong S & Whitehouse CM (1989) Science 246, 64–71). Today, with the mass spectra of intact ribosomes at 2.3 MDa becoming almost routine and the first electrospray spectra of membrane-embedded motors being recorded recently, new challenges are emerging. Knowledge of the intact mass of a protein or complex is only part of the MS information available. Data from the disruption of protein complexes in solution and gas phases are leading to subunit interaction maps and architectural models. Such models are enhanced by coupling with ion mobility in which the collision cross-section of a protein complex can be defined. Linking these attributes with knowledge of subunit dynamics and the role of post-translational modifications on the stability and interactions within complexes is increasing our understanding of the factors that stabilize and convert protein complexes between different quaternary states. From our earliest experiments, studying the folding of individual proteins, through to the characterization of membrane-embedded motors, it is clear that the full potential of electrospray in structural biology has yet to be realized. The present review offers a personal view of the transition from determining the mass of an individual protein to elucidating the structure and dynamics of heterogeneous assemblies in the megadalton mass range.

Introduction MS, long the tool of the analytical chemist, was transformed almost two decades ago subsequent to the discoveries of two ionization techniques: electrospray [1] and matrix-assisted laser desorption [2,3]. With the development of these two ionization methods, it became possible to transmit intact proteins and nucleotides into

the gas phase of the mass spectrometer, without chemical modification. Together, these two developments advanced MS from the toolbox of the analytical chemist to the research instrument of the biologist. In the post genomic era, it is not surprising that these ionization techniques have undergone further

Abbreviations CMC, critical micelle concentration; CTAB, cetyltrimethylammonium bromide; EM, electron microscopy; HSP, heat shock protein.

FEBS Journal 279 (2012) 663–677 ª 2011 The Author Journal compilation ª 2011 FEBS

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development and found subsequent application in the vast area of proteomics [4]. For these applications, proteins are typically digested into peptides, which are then used as sequence tags to identify proteins and to locate post-translational modifications. With the advent of quantitative proteomics, protein levels in response to stress or other stimuli can be addressed, providing remarkable insight into a number of disease states [5]. In parallel with these developments in proteomics, there has been a steady growth in the number of applications of electrospray to structural biology. Many researchers have made excellent contributions to this area over the past two decades. Particularly at the outset, the laboratories of Jack Henion [6], Brian Chait [7], Ken Standing [8], David Smith [9] Richard Smith [10] and Joseph Loo [11], as well as more recently Albert Heck [12], Alison Ashcroft [13] and Mike Bowers [14], have contributed greatly to this research subject. Without doubt, these researchers have advanced the field significantly. Because this is an Invited Review, associated with my lecture (FEBS ⁄ EMBO Women in Science lecture), what follows is a personal account of the developments in my laboratory over the past two decades.

Protein folding: from single proteins to first complexes Our earliest interests in electrospray came from investigations of the protein-folding problem in which researchers were trying to establish whether all proteins fold via the same route, through partially-folded, intermediate states or directly to the native state [15]. To investigate the folding pathway of individual proteins, we developed a protocol whereby all labile hydrogens were exchanged with deuterium and unfolded in deuterated chemical denaturant. Dilution of the denaturant initiated the folding reaction. High pH pulses of protons were used to detect deuterium in folded structure, protected from the labelling pulse. Mass spectra recorded in aqueous solvent to preserve the labelled protein (in the first instance hen lysozyme) allowed us to demonstrate the existence of parallel folding pathways in which some protein molecules proceeded directly to the native state, whereas others became trapped in partially-folded intermediate states. Having established this hydrogen exchange protocol for monitoring exchange globally, we were able to study a range of proteins and to compare folding with respect to the presence or absence of disulfide bonds [16], as well as wild-type and variants associated with amyloid diseases [17]. These early experiments prompted our interest in the role of cofactors in promoting protein folding. We 664

noted, at first with some scepticism, that the formation of folded protein coincided with cofactor binding [18]. We had not set out to preserve interactions between the protein and the acyl CoA cofactor, although the correlation between folding and binding was intriguing. Of further interest was the observation that the longer the alkyl chain of the cofactor, the less stable the complex in the gas phase. This was unexpected because, in solution, hydrophobic burial of the tail was known to contribute to the overall stability [19]. However, this was not mirrored in the gas phase of the mass spectrometer, where shorter hydrophobic chains bound with equal affinity to the longer ones [18]. This absence of a strong hydrophobic effect was one of the reasons why it was difficult to gain acceptance for studying intact protein ligand complexes in the gas phase during the early days of electrospray. It was clear, however, that monitoring protein folding, either alone or in the presence of cofactors, would inform not only on the rates of folding, but also on the presence of stable intermediates [20]. Because protein folding in the cell is known to proceed through interactions with other proteins, we investigated interactions of folding proteins with the molecular chaperone GroEL [21,22]. This 14-mer with a molecular mass of 800 kDa was known to have a role in folding, although its interactions with substrates were not well defined. During the early 1990s, we used electrospray to launch this 800-kDa complex into the mass spectrometer and used the transition from solution to gas phase to enable the release of the protein ligand from the dissociated chaperone. We then characterized the protein ligand with respect to its hydrogen-deuterium content and hence its degree of folding [22]. At the time, this complex was considered extremely large for electrospray. The premise of the experiment was that the complex did not dissociate when in the electrospray solution because this would affect its exchange properties. It was imperative that the complex remained intact until the phase transition, after which no further exchange could take place. From these experiments, we found that the ligand, within the GroEL tetradecamer, was more folded than a denatured state but less folded than the native state. These observations allowed us to conclude that it was present in a partially-folded conformer [22]. To confirm that the complex could remain intact during electrospray, we set about designing a mass spectrometer that would be able to cope with these very high masses. This led to collaboration with an instrument manufacturer aiming to build a mass spectrometer that was capable not only of transmitting high mass particles, but also could be used for their


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Fig. 1. One of the first successful macromolecular complexes transmitted through the mass spectrometer was the GroEL14-mer. Although it was not possible to separate peaks to the baseline in this early spectrum, which was recorded before installation of a high mass range quadrupole considerable, progress over the years has enabled complete resolution of the charge states, as well as confirmation of the presence of a single ring species. This spectrum not only confirms the overall stoichiometry, but also at that time highlighted to us the possibility of defining the stoichiometry and interactions within assemblies, in advance of other structural biology information.

tandem MS [23]. As such, it was necessary to incorporate a high mass quadrupole, capable of isolating m ⁄ z ratios of up to 32 000. With this instrument, we found that we were able to transmit caesium iodide clusters up to 80 000 m ⁄ z. Our first mass spectra of GroEL, however, required much faith to interpret the charge state series assigned to the tetradecamer and its singlering counterpart (Fig. 1) [24]. Nowadays, with several improvements to the design and operating procedures, the spectrum is routinely resolved to the baseline, enabling high resolution mass measurement and characterization of all species, including substrate proteins [25].

First megadalton complexes After this initial success, it was tempting to introduce ever larger complexes into the mass spectrometer to

try to define the upper limit that had eluded Fenn in 1989. Early results showed that we could maintain 180 copies of the intact MS2 bacteriophage virus particle at 2.5 MDa [26]. In hindsight, it is perhaps not so unexpected that a virus can survive the phase transition because transmission in the atmosphere is one of the natural vector mechanisms available to viruses for infection. The technique of electrospray closely mimics this mechanism by producing a fine aerosol of multiply-charged ions from solution [26]. A more challenging problem for MS than the ‘protein-only’ GroEL tetradecamer or the MS2 virus capsid was the ribosome, which, in bacteria, is 2.3 MDa in mass and composed of at least 50 different proteins and three very large RNA molecules. These particles also have a strong requirement for Mg2+ in solution, which can seriously hamper the electrospray process. Our first attempts to transmit intact ribosomes confirmed that it was possible to find conditions where the complex was maintained but resolution was far from optimal. Broad unresolved signal corresponding to the anticipated m ⁄ z region of intact Escherichia coli ribosomes could be obtained. Without the ability to assign charge states, however, it was not possible to determine masses of the 50S and 70S particles [27]. These early studies yielded details of the extent of protein dissociation in the gas phase and enabled us to relate this to the strength of interaction between the various ribosomal proteins and the RNA scaffold [28]. The first proteins to dissociate were found to be those located in the peripheral ‘stalk’. This information could be used to our advantage because their very dynamic nature often complicates their analysis by X-ray diffraction. To improve resolution, we investigated an alternative source and found that we could obtain much greater spectral quality with ribosomes from Thermus thermophilus. These ribosomes are known to be more robust than their E. coli counterparts [29]. Consequently, we were able to obtain mass spectra in which even the 70S subunit shows charge state resolution [30] (Fig. 2). Unexpectedly, these spectra demonstrated greater complexity of the stalk complex than was anticipated. At that time, all bacterial ribosomes were assumed to have pentameric stalks. Our observation of two additional copies of the L7 ⁄ L12 proteins led us to define a heptameric stalk complex. This was later also reported in the X-ray crystallography of L10 and the six N-terminal domains of L12 from Thermotoga maritime and used to model the first complete structure of a ribosome with the stalk complex attached (Fig. 2) [31]. The observation of additional copies of L7 ⁄ L12 in the ribosomal stalk complex in thermophilic species


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Fig. 2. The mass spectrum of intact ribosomes from T. thermophilus at a mass of 2.3 MDa, and with more than 50 different proteins and three large RNA molecules, represented a significant milestone in our research. The complex at 96 kDa is consistent with a heptameric stalk as opposed to the canonical pentameric stalks observed in all prokaryotic ribosomes up until this point. Subsequent X-ray analysis of the L10 and the N-terminal domain of L12 revealed the atomic structure of this interaction region and enabled modelling of this stalk in which all L12 molecules are in an extended conformation.

prompted us to study the interactions that modulate the binding L7 ⁄ L12 to L10 and also of the stalk complex to the ribosome itself. We found that phosphorylation of L12 increased the interaction between L10 and L12 [30]. Phosphorylation has also subsequently been reported for L7 ⁄ L12 in Streptomyces coelicolor [32] and for L7 ⁄ L12 in E. coli, predominantly at the N-terminus [33] consistent with their ability to promote interactions with L10. Using hydrogen exchange labelling of ribosomes, we were also able to define the role of the acetylated N-terminus in increasing interactions between the N-terminal helices of L7 and their interaction with L10 [34]. Further study of the multimeric nature of the stalk complexes across the phylogenetic tree revealed that, for the eukaryotic ribosomes used in our study, the stalk complexes are exclusively pentameric, whereas those from the extreme thermophilic bacteria are exclusively heptameric [35]. Interestingly, MS of ribosomes from three archaeal mesophiles showed the co-existence of two populations of ribosomes with either two or three L12 dimers attached to L10 [35]. Together, these modifications, the phosphorylation, acetylation and the copy number, appear to fine tune ribosomes for different cellular conditions. The heterogeneity and modifications of the stalk proteins prompted a key question. If these modifications affect stability, how does the composition of the stalk change because it is known that there is no conversion between L7 and L12 via acetylation or deactelyation [36]? It is likely that the ribosome is able to modify the composition of the stalk complex by 666

exchange of proteins in the cell. To investigate this possibility, we carried out subunit exchange reactions in which ribosomes uniformly labelled with 13C and 15 N were mixed with ribosomes containing natural abundance isotopes (Carol Robinson, unpublished results). The results obtained showed that exchange occurs via both dimers and monomers. Accordingly, we proposed that free L7 ⁄ L12 proteins participate in exchange with ribosome-bound L7 ⁄ L12, analogous to the situation in yeast [37], to modify the composition of the stalk. Because L12 binds more tightly to the ribosome than L7 [34], this enables ribosomes to finetune interactions within the stalk complex and to provide a strategy for survival under conditions of stress.

Unravelling heterogeneity and polydispersity In parallel with our interests in ribosomes, we pursued our early investigations into molecular chaperones by probing mechanisms of their assembly, their heterogeneity and their dynamics, as well as their thermal stability. In the first of our assembly reactions, we monitored the assembly of the archaeal version of the chaperone GimC, which cooperates with TRiC in vivo. Using the recombinant version of two subunits, a and b, which together comprise the Methanobacterium thermoautotrophicum GimC hexamer, we monitored the assembly of the complex as a function of time. The mass spectra recorded throughout the time course showed the absence of any significantly populated


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Fig. 3. Polydispersity of the alphaB-crystallin is revealed in the MS ⁄ MS analysis of the peak at 10000 m ⁄ z. Peaks corresponding to the charge states of doubly stripped oligomers are expanded and shown in comparison with a series of simulated spectra. The overlapping peak at 20 080 m ⁄ z results from oligomers carrying the same number of charges as subunits. The groups of peaks at lower m ⁄ z than this central peak result from oligomer populations carrying more than one charge per subunit, whereas those at higher m ⁄ z arise from oligomers carrying less than one charge per subunit. The grey spectrum represents the sum of the simulated spectra and is compared with the experimental data immediately below (black). This deconvolution reveals the relative intensity of the many different components.

intermediates, demonstrating that the assembly process is highly cooperative [38]. We also examined the thermal stability of the complex using a heated nanoflow probe [39]. We found that a significant proportion of the MtGimC hexamer remains intact under low-salt conditions at elevated temperatures. This result was encouraging; although only a relatively simple complex with just two different subunits, it demonstrated a proof of principle that the dynamics of assembly and the thermal stability of a molecular chaperone could be obtained from electrospray up to temperatures of 60 C. Many molecular chaperones, however, are not monodisperse like MtGimC and GroEL; their polydispersity has long been a considerable obstacle to their study. Our first spectra of the a crystallins presented a significant challenge with respect to their interpretation. The many overlapping charge states could not be

assigned unambiguously. Simulation of the individual spectra was required to reveal the precise composition of the ensemble of oligomeric states (Fig. 3). The results showed that the major oligomeric form was a 28-mer [40]. Further investigation of this complex showed that the a B crystallins had a distinct preference for even-numbered oligomers, consistent with a dimeric substructure [41]. We were also able to show that populations of different oligomers are affected either by post-translational modification [41] or truncation [42]. These early experiments demonstrated a means of probing polydisperse systems that was unique to MS and, moreover, they highlighted ways of probing the response of these ensembles to post-translational modification. The next key step in this research was to define interactions of these polydisperse molecular chaperones with substrates or client proteins. We investigated this


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by examining the dynamics of complexes formed between an oligomeric small heat shock protein (HSP) and a client protein luciferase [43]. We observed over 300 different stoichiometries of interaction, demonstrating the remarkable heterogeneity that has rendered these complexes intractable by conventional structural biology approaches. This polydispersity gives the system a behaviour that is quite distinct from the ATP-dependent chaperones, such as the GroEL system described above and the HSP90 system outlined below, which have defined stoichiometries of interaction. At first glance, HSP90, which exists in either monomeric or dimeric forms, would appear straightforward to study by MS. However, HSP90 forms heteromeric complexes by virtue of its reaction with numerous clients and cochaperones that are essential for the formation of a functional complex [44]. We set out to study this reaction cycle by investigating the addition of human HSP90 to the co-chaperones, Hop, a peptidylprolyl isomerase FKBP52 and HSP70. We monitored the formation of a predominant ternary complex containing both cochaperones (HSP90:Hop:FKBP52) [45].

This so called ‘asymmetric complex’ is subsequently able to interact with the chaperone HSP70 to form quaternary complexes containing all four proteins with differing stoichiometries. This is the functional complex that is able to bind to client proteins. Monitoring the population of these complexes, both during their formation and at equilibrium, allows us to extract 14 different KD values (Fig. 4). The KD values that we determined afford insights into the assembly of ten HSP90-containing complexes and, because all of the KD values are in the nanomolar range, which is close to their established cellular concentrations [46], this provides a rationale for the cellular heterogeneity and prevalence of intermediates in the HSP90 chaperone cycle.

Moving from recombinant to cellular complexes The study of recombinant complexes enabled us to establish a number of criteria necessary to transmit and maintain intact macromolecular complexes and

Fig. 4. (A) MS recorded from t = 0–45 min showing the formation of the asymmetric complex containing both Hop and FKBP52. (HSP90)2(Hop)1 and (HSP90)2(Hop)2 were formed with a two-fold excess of Hop before the addition of FKBP52. Charge states are coloured as: Hop (light green); HSP90 monomer and dimer (grey and black, respectively); (HSP90)2(Hop)1 (dark green); (HSP90)2(Hop)2 (purple); (HSP90)1(FKBP52)1 (yellow); (HSP90)2(FKBP2)1 (blue); (HSP90)2(Hop)1(FKBP52)1 (red). (B) An interaction network for HSP90, HSP70, Hop and FKBP52. The KD value for Hop, (HSP90)2 and FKBP52 was determined from multiple spectra and from eight different experimental data sets, (green background).The starting point for these experiments was the determination of the KD for the HSP90 equilibrium (green solid line). Favourable KD values are represented by thicker arrows. Addition of HSP70 to the ternary (HSP90)2(Hop)n(FKBP52)1 complexes leads to the formation of quaternary complexes with favourable KD values (blue background). Complexes likely to form, given the cellular concentrations of the four protein components, are circled (dotted line).

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also to probe their overall topology, assembly reactions and stability. The real test of a structural biology tool, however, lies in its ability to predict the subunit architecture of a complex before other 3D information becomes available. Once we had established that we could extract protein complexes directly from cells, at natural expression levels, as well as record mass spectra that are informative both in terms of post-translational modification, the masses of subunits and their copy number, we were in a position to tackle complexes resistant to study by other structural biology approaches [47]. Using a tap tag approach, we isolated the yeast RNA exosome, for which there was no X-ray structure at that time, and recorded the electrospray spectra of the intact complex, confirming the presence of all ten subunits at unit stoichiometry. Interestingly, homology modelling had predicted a series of interactions [48], although these were not in-line with previous yeast two-hybrid studies [49]. We therefore decided to define interactions within this complex ourselves using a series of solution disruption experiments. These experiments led to the generation of three pairwise dimers that form the ring [47]. A number of trimers and tetramers allowed the definition of the bridging subunits that confer stability to the ring, and the base subunit was located according to its ability to stabilize one side of the ring. With the subsequent publication of the X-ray structure of the human exosome [50], cryo-electron microscopy (EM) structures of the yeast exosome [51,52] and X-ray structures of subcomplexes [53], this predictive model was found to be correct in terms of all subunit interactions and overall architecture. Later, we developed this subunit interaction model into an atomic model via homology modelling [54]. Because the atomic structure of the yeast exosome still remains unavailable, this model serves as an example of the power of combining homology modelling with interactions derived from MS for producing atomic models of complexes that are too dynamic or heterogeneous for study by X-ray crystallography. Encouraged by this combination of defining protein interactions through disassembly in solution and defining the composition of the building blocks, we tackled a more challenging system, the eukaryotic initiation factor 3, which binds to the 40S subunit of the eukaryotic ribosome and also to internal ribosome entry site viruses. Proteomic investigations had identified 13 subunits together with numerous post-translational modifications [55]. Placing these 13 subunits within the EM density map [56], however, was not possible because the full set of interactions was unknown. Using MS of the intact complex, we were able to establish the copy number and homogeneity of the complex. Gener-


ating a total of 27 subcomplexes by manipulating the ionic strength of the solution, and confirming their composition through tandem MS and using internal ribosome entry site binding to distinguish which of the 13 subunits are affected by binding to RNA, we derived a 3D subunit interaction map [57]. Although it was tempting at this stage to align this complex within the 3D cryo-EM density, it was clear that there was insufficient information to achieve this. Additional restraints were required to define the topology of the subcomplexes and to align them within the EM density map. For this, we turned to ion mobility MS.

Defining the overall topology of protein complexes Although MS as a means of determining the composition of protein complexes was becoming accepted [58], their overall topology in the gas phase remained elusive. If subunit interactions were being maintained, however, it was likely that the overall shape would bear some resemblance to that established crystallographically or in solution. This had always been difficult to prove. Ion mobility MS, which had been used for some time to probe peptide [59] and individual protein conformations [60], offered the possibility of obtaining a first glimpse of the topology of a protein complex in the gas phase. Choosing a well-defined ring shape structure, TRAP (tryptophan RNA attenuation binding protein), we reasoned that it was likely to yield large differences between its native-like ring and its collapsed state. We used a prototype instrument and found that different charge states of the apo protein complex either collapsed or maintained ring-like structures in the gas phase [61]. In solution, the complex was known to be stabilized by the addition of tryptophan and synthetic RNA. We also found that this stability was maintained when the ternary TRAP:RNA:tryptophan complex was electrosprayed into the ion mobility-MS. Moreover, the ring-like structure of the ternary complex could be preserved more readily than the apo form, highlighting the possibility of using this approach to probe alternative conformations of protein complexes in the gas phase. However, we first had to establish how general this would be for intact complexes and whether the collision cross-section of subcomplexes generated in solution could also be used to construct the overall topology of an assembly. To establish whether or not this would be a general phenomenon for protein complexes in the gas phase, we selected two 12-mer protein assemblies (i.e. ornithine carbamoyl transferase and glutamine synthetase) with different topologies and building blocks and


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examined both the intact complex and subcomplexes generated in solution. We found that, for the cage-like 12-mer ornithine carbamoyl transferase formed from three trimers, the collision cross-sections of the 3-mer, 6-mer and 9-mer and 12-mer matched well the modelled values from the X-ray coordinates. For glutamine synthetase, formed from two six-membered rings of dimers, the overall topology of the 12-mer was in good agreement with the native-like topology but showed some collapse of the incomplete ring, particularly for the higher oligomers [62]. The results obtained, although reinforcing the ability of ion mobility to capture the overall topology, hinted at a cautionary note: some complexes, particularly those with relatively large incomplete assemblies, may collapse in the gas phase. Despite this caution with respect to studying incomplete rings and large assemblies, the trimeric building blocks generated from the ornithine carbamoyl transferase 12-mer recapitulated the simple geometry expected from a close packed trigonal arrangement of subunits, and suggested that this ion mobility-MS approach could be applied to deduce the arrangement of subunits within subcomplexes. With the observation that the topology of trimeric building blocks could be preserved in the gas phase, we returned to an early problem: that of the eukaryotic initiation factor 3. We had established in our study of 27 subcomplexes that we could form two trimers: e:l:k and f:h:m [57]. We suspected that one had a linear arrangement of subunits, whereas the other was likely to be compact. Using ion mobility measurements to obtain collision cross-sections and by comparing these with coarse grained models, we were able to distinguish the close-packed f:h:m form the linear e:l:k [62] and to fit these complexes into distinct appendages on the EM density map. With the subunit connectivity, overall topology and shape of the building blocks of protein complexes becoming established, we turned our attention to perhaps our ultimate challenge to date: that of transmitting an intact membrane protein complex into the gas phase.

The structural properties of gas phase micelles Returning again to the problems of maintaining strong hydrophobic effects that first concerned us in 1996, with the acyl CoA binding protein, we began a systematic study of protein detergent complexes. Our initial attempts to obtain mass spectra of these complexes were thwarted, primarily as a result of the large excess of detergent molecules necessary to solubilize membrane complexes. This had a deleterious effect on the 670

mass spectra, leading to a high background of detergent clusters and compromising our ability to observe any peaks for protein components. Despite these difficulties, some progress was made with peripheral membrane proteins. In particular, the mass spectra of apolipoprotein C-II, with heterogeneous suspensions of phospholipids, demonstrated that the protein can bind simultaneously to two different phospholipids [28]. Moreover, when apolipoprotein C-II is added to lipid suspensions with immiscible lipids, the protein is capable of remodelling the distribution to form one that is closer to a statistical arrangement. During this study, we also found that we were able to maintain associations of phospholipid clusters containing in excess of 80 molecules [28]. The observation of these detergent clusters raised our interest in studying proteins within micelles, selecting as our first example the simple protein dimer EmrE binding to the ligand TPP+ [63]. The peaks assigned to submicelles, or detergent aggregates, were very broad in nature and consistent with a heterogeneous distribution of detergent molecules adhering to the protein complex. As such, the mass spectrum could not be interpreted [64]. To simplify this complexity, we used a tandem MS procedure in which discrete m ⁄ z values were isolated from the broad peaks and subjected to collision induced dissociation. These spectra reveal clusters of dodecylmaltoside molecules, as well as the sequential release of the ligand TPP+ and monomeric EmrE as the collision cell voltage was raised [64]. The fact that this membrane protein ligand complex could survive in the gas phase was encouraging, although at that stage, charge states could not be assigned to the protein dimer. However, the results implied that aspects of the micellar structure were stable in the gas phase. It was not clear how much of the assembly could be maintained, whether these detergent assemblies inverted in the gas phase, and what factors governed their overall stability. To address the properties of detergents in the gas phase, we studied micelles, reverse micelles and reverse micelles encapsulating myoglobin using the same surfactant (cetyltrimethylammonium bromide; CTAB) and manipulated the aqueous and organic phases to form normal and reverse micelles [65]. Applying tandem MS revealed differences in the ions that dissociate. Those that dissociate from regular micelles have undergone > 90% exchange of bromide ions from the headgroup with acetate ions from the aqueous solvent. By contrast, for reverse micelles, ions are detected without exchange of bromide ions from the headgroup, consistent with their protection in the core of the micellar structure. This demonstrates that the reverse micelle structure is retained in the electrospray droplet.


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Once in the gas phase, tandem mass spectra of micelles and reverse micelles revealed polydisperse assemblies containing several hundred CTAB molecules [65]. These numbers were much greater than those predicted from aggregation numbers determined in solution ( 90), indicating the coalescence of the micellar systems to form large assemblies. For reverse micelles incorporating myoglobin, spectra were consistent with one holo myogolobin molecule in association with 270 CTAB molecules. This compares well with an estimation of the number of molecules required to fully encapsulate myoglobin based on its surface area and the cross-sectional area of the CTAB headgroup ( 200 CTAB molecules) [65]. The increase in the number determined experimentally could be attributed to the contribution of ordered water molecules to the surface area of the encapsulated protein. Overall, these results showed that, although the solution-phase orientation of surfactants is preserved during electrospray, the numbers of detergent molecules in regular micelles do not necessarily coincide with those that would be predicted from solution phase studies of micelles. Following on from these observations, and in an attempt to understand further the properties of micelles, we aimed to establish a correlation between their behaviour in the gas phase with solution phase properties (Carol Robinson, unpublished results). We employed ion mobility-MS to separate and assign detergent clusters formed from a series of six n-trimethyl ammonium bromide detergents with different alkyl chain lengths. We showed that the number of ntrimethyl ammonium bromide molecules in a cluster is independent of the detergent concentration in solution and increases with charge state but, unexpectedly, decreases with alkyl chain length. This relationship cannot therefore be explained by the factors that govern micelle formation in solution where alkyl chain length is a major determinant [66]. Employing a liquid drop model, which considers both the surface energy and charge, however, correlates extremely well with the experimental cluster size. This understanding of the role of charge with respect to stability, as well as the importance of hydrophobic tail in governing stability, informed our choice of detergents for the subsequent study of membrane protein complexes.

Membrane protein complexes at last With the beginnings of an understanding of the factors that govern micelle stability and the hint that the membrane protein EmrE could be transported into the gas phase came the growing realization that it might become possible to observe intact membrane com-


plexes provided that we selected the appropriate detergent, with respect to chain length and charge, and kept the detergent concentration above the critical micelle concentration (CMC) to maintain solubility in the electrospray droplet. Our first success was that of the heterotetramer, the vitamin B12 importer BtuC2D2, which contains two cytoplasmic (BtuD) and two transmembrane subunits (BtuC) [67]. By introducing this complex from a dodecylmaltoside solution above the CMC and activation in the gas phase, we were able to maintain both cytoplasmic and membrane protein interactions, at the same time as removing the detergent micelle [68]. This not only revealed the tetrameric structure, but also enabled us to probe the effects of nucleotide binding. This was a significant advance because, until this time, it had been widely acknowledged that predominantly hydrophobic interactions would have little or no chance of survival in the gas phase. However, the activation energy required to release this complex from the micelle was considerably larger than would be used for a soluble complex of similar mass [69,70], not only highlighting the protection afforded by the micelle, but also prompting the next key question. Because it is established that increases in protein collision cross-section result from increasing internal energy [71], if the protein micelle complex was accelerated into the collision cell to remove detergent from the protein complex, was the energy sufficient to initiate unfolding of proteins within the complex? To investigate this possibility, we selected two tetrameric membrane complexes of similar mass and surface area: BtuC2D2 [67], as used above to demonstrate feasibility, and KirBac3.1, which has different subunit packing. In the latter tetramer, the ion channel, all four subunits make contact with the membrane. We used ion mobility-MS to address the question above and demonstrated that both membrane protein complexes could be maintained in relatively compact states after their release from detergent micelles [72] (Fig. 5). We also found that the quaternary structure of the transporter, which has fewer transmembrane subunits than the ion channel, is less stable in the gas phase, with a BtuC subunit unfolding and dissociating first. These results highlight the potential of ion mobility-MS for characterizing the overall topologies of membrane protein complexes and suggest future applications whereby structural changes could be associated with nucleotide, lipid and drug binding. With this change in thinking, suggesting that it was becoming possible to maintain hydrophobic interactions, we began to investigate a large number of membrane protein complexes from simple dimers that


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Fig. 5. (A) Ion mobility-MS of the Kirbac3.1 tetramer (Protein Data Bank code: 1XL6) and (B) the BtuC2D2 tetramer (Protein Data Bank code: 1L7V). The four subunits are coloured pink, yellow (BtuD), blue and green (BtuC). Black planes indicate the hydrophobic boundaries of the transmembrane regions. Insets: Arrival-time distributions for the 10+ charge state of trimeric KirBac3.1 and BtuCD2 formed via gas phase dissociation of the respective tetramers, superimposed on the collision cross-section axis. Representative structures and calculated collision cross-section (blue dotted lines) of a compact Kirbac3.1 trimer (a); as in the inset structure (a) but with collapsed N-and C-termini (b); a BtuCD2 trimer with a three-fold symmetric structure with collapsed N-termini (c); as in (c) but with partial unfolding of BtuD (d); and the tetrameric structure with collapsed N-termini and a BtuC subunit removed but without three-fold symmetry (e).

form in the membrane through to very large macromolecular machines. Revisiting the simple dimer EmrE, we found that, under conditions under which the CMC of detergent was exceeded, the dimer was preserved [69]. One of the surprising aspects of this research was the observation that, irrespective of the size of the complex, in almost all cases, a selection of lipids remained tightly bound to the membrane protein complex. For the two ABC transporters MacB and LmrCD, found to be almost exclusively homodimeric 672

and heterodimeric additional peaks could be assigned to one and two molecules of phosphatidylethanolamine and cardiolipin in the case of MacB and LmrCD dimers, respectively. Further experiments, however, showed that the bound lipids were largely invariant of the detergent used for their extraction, and in many cases, remained attached to the protein even after collision induced dissociation. This must mean that they are binding through a specific interaction, not readily disrupted by collisions. A similar finding became one of the defining moments in our analysis of the two rotary V-type ATPases of T. thermophilus and Enterrococcus hirae both of which have very different lipid binding properties [73]. Despite the fact that the mass spectra of these two membrane motors were relatively well–resolved, we were unable to assign these spectra for a number of reasons (Fig. 6). Primarily, the presence of a number of tight binding lipids was unexpected at that time and led us to consider that additional subunits were present. After dissociation of the complexes, measurement of the mass of the individual subunits and characterization of the lipid components, we realized that multiple lipids were attached to the membrane rings, leading us at first to wrongly assign their subunit stoichiometry. These discrepancies were revealed by a software package designed in-house [74]. The software package employed is able to assign variable mass shifts to membrane and soluble subunits and also able to simulate spectra for complexes of many possible stoichiometries and subunit compositions, ultimately producing an optimal fit between the raw data and the sum of the component spectra. With this software in place, we could then identify not only sub-stoichiometric binding of lipids, but also nucleotides. These small molecules turned out to be the key to unlocking some of the functional aspects of these molecular motors (Fig. 6). The partial binding of lipids in the rotary ring enabled us to propose a change in the topology of the 12-membered ring to a six-fold symmetric structure stabilized by six lipids binding between pairs of subunits within the rotor ring. Nucleotide binding to the head and the base induced changes in subunit interactions in the head and, unexpectedly, also in the VO complex. Using ion mobility, we demonstrated enhanced conformational dynamics of subunit I, which contains both membrane and soluble domains. This led us to propose a new regulatory model, in which the soluble region of subunit I binds more tightly to ADP sensing depletion of ATP, signalling its dissociation. Subsequently, membrane lipids take the place of subunit I and, consequently, close the proton channel. This study therefore encompasses many of the


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Fig. 6. Main panel mass spectrum of the intact T. thermophilus ATPase released from a dodecylmaltoside micelle. The intact species gives rise to a series of well-resolved charge states (purple stars). Loss of subunit E in the gas phase yields a stripped complex (blue circles). The membrane-embedded complex ICL12 dissociates in solution (26+ series) and in the gas phase (34+ series). (A) Deconvolution of the mass spectrum recorded for the membrane-embedded ICL12 complex reveals the contributions from gas phase dissociation (orange); solution phase disassembly (green); and adjustment of intensity and summation to form the simulated spectrum (blue) for comparison with the raw data (black), which was performed using a software package designed in-house [74]. The assignment of the masses of these peaks revealed six lipid and one or two nucleotide binding sites. (B) Model of the membrane-embedded rotor (the ICL12 complex) showing the six lipid binding sites in yellow, the L12 subunit in red, subunit C (blue) and a model for subunit I (grey). Ion mobility-MS of the membrane-embedded rotor revealed enhanced dynamics of subunit I in response to nucleotide binding (proposed nucleotide binding site; shown in orange). This ADP binding site is in accordance with findings for a eukaryotic equivalent of subunit I [80]. We proposed, based on ion mobility measurement of collision cross-section, that subunit I flexes at the hinge region, inducing its dissociation from the ring and hence a disruption of the proton channel (C). Tandem MS (not shown here) was used to demonstrate the attachment of subunit F to the soluble head, preventing rotation of the central shaft, thereby preventing its unregulated ATP consumption (D).

strategies that we have been working towards for some time. These comprise the ability to assign large heterogeneous multicomponent complexes of unknown stoichiometry after isolation form cells, an understanding of the disassembly of the complexes in solution and gas phases, the role of small molecules in changing interactions, and the application of ion mobility to constrain molecular models of macromolecular assemblies. With these developments in place, we have almost reached all of our original goals. However, one further challenge has remained elusive for almost a decade: the delivery of an image of an electrosprayed protein complex after its flight in the gas phase.

Seeing is believing Our ability to transport assemblies large enough to see in an electron microscope stimulated our interest in

trying to capture these particles to show how they are affected by their flight. We were also intrigued by the possibility of using an MS–EM combination in a preparative way, aiming to isolate complexes with a given m ⁄ z value, and hence to provide homogeneous specimens for imaging. With this challenge in mind, we designed an EM grid to sit in the path of the ion beam in a mass spectrometer, for collection of particles after the separation of gas phase ions in the high mass quadrupole. Almost full circle, we went back to our original complex, namely that of GroEL, on which we had optimized the passage of macromolecular ions some 10 years earlier. Using a holey carbon grid, we captured the protein complex during various stages of its flight in the mass spectrometer [75]. Immediately after electrospray, the particles were readily identified on the EM grid. After the quadrupole mass filter, collection on the grid was more difficult because steering


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voltages and lenses had to be optimized to ensure a soft landing. Our first images were consistent with the collection of GroEL on the grid but not of sufficient quality to define structural changes were a result of these processes [75]. Further optimization of our deposition energies and our mass selection from heterogeneous mixtures of particles is leading to better control and clearer images.

Future directions Returning now to the absence of an upper mass limit, as first predicted by Fenn, it would have been impossible to foresee the extent to which electrospray has been involved in the ionization of intact macromolecular assemblies of increasing size and complexity. The rise in the upper mass limit has been demonstrated many times through the study of high molecular mass ribosomes [30], virus capsids [26] and intact viruses up to 30–40 MDa [76,77]. These achievements prompt new challenges. That the topology and packing of subunits can now also be revealed from the electrospray of complexes after appropriate treatment in solution or gas phases opens the way to assembling models of the 3D subunit architecture. As a standalone approach, MS, together with constraints from ion mobility measurements, can now be used to propose 3D architectural models. It is of course much more powerful when used in combination with other methods in hybrid structural biology approaches, in particular with EM, X-ray and NMR [78,79]. In closing, this move from mass to structure heralds a new era for electrospray; one that is even more distant from the analytical tool for which it was once established. In the same way that it has not been possible to predict the upper mass limits for electrospray, perhaps it would be even more foolish to try to predict the future roles of electrospray in structural biology. It is hoped that capturing the heterogeneity and dynamics of membrane and soluble complexes, as close as possible to their cellular environment, will become accepted as a major force in structural biology. As such, it is hoped that it will stimulate researchers to generate new hypotheses for the cellular modes of operation of macromolecular complexes for many years to come.

Acknowledgements I am grateful to all past and present members of my research group for their commitment and enthusiasm for this research. I am particularly grateful for the critical review from Helena Hernańdez, Nina Morgner 674

and Justin Benesch. I acknowledge with thanks funding from the Royal Society, an ERC advanced investigator award, The Wellcome Trust, the MRC, the BBSRC and the EU.

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66 Hamada N & Einaga Y (2005) Effects of hydrophobic chain length on the characteristics of the micelles of octaoxyethylene tetradecyl C(14)E(8), hexadecyl C(16)E(8), and octadecyl C(18)E(8) ethers. J Phys Chem B 109, 6990–6998. 67 Locher KP, Lee AT & Rees DC (2002) The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Science 296, 1091–1098. 68 Barrera NP, Di Bartolo N, Booth PJ & Robinson CV (2008) Micelles protect membrane complexes from solution to vacuum. Science 321, 243–246. 69 Barrera NP, Isaacson SC, Zhou M, Bavro VN, Welch A, Schaedler TA, Seeger MA, Miguel RN, Korkhov VM, van Veen HW et al. (2009) Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions. Nat Methods 6, 585–587. 70 Barrera NP & Robinson CV (2011) Advances in the mass spectrometry of membrane proteins: from individual proteins to intact complexes. Annu Rev Biochem 80, 247–271. 71 Benesch JL, Ruotolo BT, Simmons DA & Robinson CV (2007) Protein complexes in the gas phase: technology for structural genomics and proteomics. Chem Rev 107, 3544–3567. 72 Wang S, Politis A, Di Bartolo N, Bavro V, Tucker S, Booth P, Barrera N & Robinson C (2010) Ion mobility mass spectrometry of two tetrameric membrane protein complexes reveals compact structures and differences in stability and packing. J AmChem Soc 132, 15468–15470. 73 Zhou M (2011) Mass spectrometry of intact V-type ATPases reveals lipid binding and the effects of nucleotide binding. Science 344, 380–385.


74 Hernańdez H, Makarova OV, Makarov E, Muto Y, Pomeranz-Krummel D & Robinson CV (2009) Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1snRNP. PLoS One 4, e7202. 75 Benesch JL, Aquilina JA, Baldwin AJ, Rekas A, Stengel F, Lindner RA, Basha E, Devlin GL, Horwitz J, Vierling E et al. (2010) The quaternary organization and dynamics of the molecular chaperone HSP26 are thermally regulated. Chem Biol 17, 1008–1017. 76 Fuerstenau SD, Benner WH, Thomas JJ, Brugidou C, Bothner B & Siuzdak G (2001) Mass spectrometry of an intact virus. Angew Chem Int Ed Engl 40, 9822. 77 Uetrecht C, Versluis C, Watts NR, Roos WH, Wuite GJ, Wingfield PT, Steven AC & Heck AJ (2008) High-resolution mass spectrometry of viral assemblies: molecular composition and stability of dimorphic hepatitis B virus capsids. Proc Natl Acad Sci USA 105, 9216–9220. 78 Robinson CV, Sali A & Baumeister W (2007) The molecular sociology of the cell. Nature 450, 973–982. 79 Baldwin AJ, Lioe H, Robinson CV, Kay LE & Benesch JL (2011) AlphaB-crystallin polydispersity is a consequence of unbiased quaternary dynamics. J Mol Biol 413, 297–309. 80 Armbruster A, Hohn C, Hermesdorf A, Schumacher K, Borsch M & Gruber G (2005) Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding. FEBS Lett 579, 1961–1967.


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