Twenty Years of Gas Phase Structural Biology
2013; Elsevier BV; Volume: 21; Issue: 9 Linguagem: Inglês
10.1016/j.str.2013.08.002
ISSN1878-4186
AutoresJulien Marcoux, Carol V. Robinson,
Tópico(s)Protein Structure and Dynamics
Resumo•History of the development of mass spectrometry for membrane protein complexes•Progress from the first mass spectra to the effects of PTMs and lipid binding•Future perspectives including enhanced resolution of mass spectra and ion mobility Over the past two decades, mass spectrometry (MS) of protein complexes from their native state has made inroads into structural biology. To coincide with the 20th anniversary of Structure, and given that it is now approximately 20 years since the first mass spectra of noncovalent protein complexes were reported, it is timely to consider progress of MS as a structural biology tool. Early reports focused on soluble complexes, contributing to ligand binding studies, subunit interaction maps, and topological models. Recent discoveries have enabled delivery of membrane complexes, encapsulated in detergent micelles, prompting new opportunities. By maintaining interactions between membrane and cytoplasmic subunits in the gas phase, it is now possible to investigate the effects of lipids, nucleotides, and drugs on intact membrane assemblies. These investigations reveal allosteric and synergistic effects of small molecule binding and expose the consequences of posttranslational modifications. In this review, we consider recent progress in the study of protein complexes, focusing particularly on complexes extracted from membranes, and outline future prospects for gas phase structural biology. Over the past two decades, mass spectrometry (MS) of protein complexes from their native state has made inroads into structural biology. To coincide with the 20th anniversary of Structure, and given that it is now approximately 20 years since the first mass spectra of noncovalent protein complexes were reported, it is timely to consider progress of MS as a structural biology tool. Early reports focused on soluble complexes, contributing to ligand binding studies, subunit interaction maps, and topological models. Recent discoveries have enabled delivery of membrane complexes, encapsulated in detergent micelles, prompting new opportunities. By maintaining interactions between membrane and cytoplasmic subunits in the gas phase, it is now possible to investigate the effects of lipids, nucleotides, and drugs on intact membrane assemblies. These investigations reveal allosteric and synergistic effects of small molecule binding and expose the consequences of posttranslational modifications. In this review, we consider recent progress in the study of protein complexes, focusing particularly on complexes extracted from membranes, and outline future prospects for gas phase structural biology. The Nobel Prize for chemistry was awarded in 2002 for the development of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), the so-called soft ionization methods that together revolutionized protein characterization. These ionization methods have led to the widespread use of mass spectrometry (MS) in all aspects of the life sciences. Over the past 20 years, MS has spawned entirely new research fields, including proteomics and metabolomics, and created new combinations, such as the use of MS with MALDI imaging (Norris and Caprioli, 2013Norris J.L. Caprioli R.M. Analysis of tissue specimens by matrix-assisted laser desorption/ionization imaging mass spectrometry in biological and clinical research.Chem. Rev. 2013; 113: 2309-2342Crossref PubMed Scopus (494) Google Scholar). For proteomics, enzymatic digestion is used to generate peptides that can be fragmented to obtain the amino acid sequence, enabling proteins to be identified from databases (Shevchenko et al., 1996Shevchenko A. Jensen O.N. Podtelejnikov A.V. Sagliocco F. Wilm M. Vorm O. Mortensen P. Shevchenko A. Boucherie H. Mann M. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels.Proc. Natl. Acad. Sci. USA. 1996; 93: 14440-14445Crossref PubMed Scopus (1298) Google Scholar). In a recent impressive example, protein expression levels and functionalization through posttranslational modifications (PTMs) were deduced for more than 6,500 predicted proteins of Trichoplax adhaerens (Ringrose et al., 2013Ringrose J.H. van den Toorn H.W. Eitel M. Post H. Neerincx P. Schierwater B. Altelaar A.F. Heck A.J. Deep proteome profiling of Trichoplax adhaerens reveals remarkable features at the origin of metazoan multicellularity.Nat. Commun. 2013; 4: 1408Crossref PubMed Scopus (39) Google Scholar). In addition to primary sequence determination, when coupled to a wide range of labeling approaches, MS can also inform on the higher-order structure and dynamics of proteins and their complexes. These labeling approaches include hydrogen-deuterium exchange (Landreh et al., 2011Landreh M. Astorga-Wells J. Johansson J. Bergman T. Jörnvall H. New developments in protein structure-function analysis by MS and use of hydrogen-deuterium exchange microfluidics.FEBS J. 2011; 278: 3815-3821Crossref PubMed Scopus (22) Google Scholar, Wales et al., 2013Wales T.E. Eggertson M.J. Engen J.R. Considerations in the analysis of hydrogen exchange mass spectrometry data.Methods Mol. Biol. 2013; 1007: 263-288Crossref PubMed Scopus (46) Google Scholar), hydroxyl radical footprinting (Konermann and Pan, 2012Konermann L. Pan Y. Exploring membrane protein structural features by oxidative labeling and mass spectrometry.Expert Rev. 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Limited proteolysis coupled to tandem MS can also be used to locate accessible residues for structural elucidation or to improve crystallogenesis (reviewed in Cohen and Chait, 2001Cohen S.L. Chait B.T. Mass spectrometry as a tool for protein crystallography.Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 67-85Crossref PubMed Scopus (56) Google Scholar). Recent highlights include hydrogen-deuterium exchange experiments, used to define changes in the interaction interface of the heterotrimeric bovine G protein, upon binding of the agonist-bound human β(2) adrenergic receptor (Chung et al., 2011Chung K.Y. Rasmussen S.G. Liu T. Li S. DeVree B.T. Chae P.S. Calinski D. Kobilka B.K. Woods Jr., V.L. Sunahara R.K. Conformational changes in the G protein Gs induced by the β2 adrenergic receptor.Nature. 2011; 477: 611-615Crossref PubMed Scopus (290) Google Scholar). Results reveal higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the complex, likely due to the dynamics of the component proteins and their interaction interface. Hydroxyl radical footprinting experiments can also report on secondary structure and importantly can be used to identify sites of bound water in cytochrome c and ubiquitin, revealing the millisecond dynamics of water molecules in protein crevices (Gupta et al., 2012Gupta S. D'Mello R. Chance M.R. Structure and dynamics of protein waters revealed by radiolysis and mass spectrometry.Proc. Natl. Acad. Sci. USA. 2012; 109: 14882-14887Crossref PubMed Scopus (37) Google Scholar). Tracking interconverting states using fast oxidative labeling was also essential to understanding the early submillisecond folding events of the barstar protein (Chen et al., 2012Chen J. Rempel D.L. Gau B.C. Gross M.L. Fast photochemical oxidation of proteins and mass spectrometry follow submillisecond protein folding at the amino-acid level.J. Am. Chem. Soc. 2012; 134: 18724-18731Crossref PubMed Scopus (86) Google Scholar). These exciting applications highlight opportunities for probing previously indefinable protein-water interactions and water dynamics within proteins and their complexes as well as very fast protein folding mechanisms. Chemical crosslinking experiments have recently come to the fore with distance restraints leading to construction of detailed models of the human protein phosphatase 2A (PP2A) complexes, linking specific trimeric PP2A complexes to adaptor proteins (Herzog et al., 2012Herzog F. Kahraman A. Boehringer D. Mak R. Bracher A. Walzthoeni T. Leitner A. Beck M. Hartl F.U. Ban N. et al.Structural probing of a protein phosphatase 2A network by chemical cross-linking and mass spectrometry.Science. 2012; 337: 1348-1352Crossref PubMed Scopus (311) Google Scholar). Interestingly, in this study, crosslinking restraints guided molecular modeling of the binding interface between immunoglobulin binding protein 1 (IGBP1) and trimeric PP2A and revealed the elusive topology of the TCP1 ring complex (TRiC) chaperonin, which interacts with a regulatory subunit 2ABG within the PP2A complex (Herzog et al., 2012Herzog F. Kahraman A. Boehringer D. Mak R. Bracher A. Walzthoeni T. Leitner A. Beck M. Hartl F.U. Ban N. et al.Structural probing of a protein phosphatase 2A network by chemical cross-linking and mass spectrometry.Science. 2012; 337: 1348-1352Crossref PubMed Scopus (311) Google Scholar). These labeling experiments are undoubtedly very powerful, particularly when used in hybrid structural biology approaches and in combination with information gleaned from the MS of intact assemblies and their subcomplexes (Robinson et al., 2007Robinson C.V. Sali A. Baumeister W. The molecular sociology of the cell.Nature. 2007; 450: 973-982Crossref PubMed Scopus (411) Google Scholar). Compared to the labeling methods described above, MS of intact complexes was initially met with more skepticism by the general structural biology community. From the earliest reports in the 1990s of proteins interacting with ligands, and first observations of protein complexes in the gas phase (reviewed in Loo, 1997Loo J.A. Studying noncovalent protein complexes by electrospray ionization mass spectrometry.Mass Spectrom. Rev. 1997; 16: 1-23Crossref PubMed Scopus (1161) Google Scholar), doubts were expressed as to the significance of these complexes given the importance placed on water in mediating the folded state of proteins (Wolynes, 1995Wolynes P.G. Biomolecular folding in vacuo!!!(?).Proc. Natl. Acad. Sci. USA. 1995; 92: 2426-2427Crossref PubMed Scopus (119) Google Scholar). Initially the contributions of hydrophobic forces to protein stability, which are absent in vacuo, were considered to be too important for protein structure to be retained. Moreover, the fact that the vacuum is an apolar hydrophobic medium led to predictions that proteins would turn inside-out in the mass spectrometer. One contribution to stability, often overlooked, is the van der Waals attraction between the residues of the polypeptide. This attractive force persists in vacuo and likely provides sufficient stability to retain subunit interactions in the gas phase. Interestingly molecular dynamics (MD) simulations have also shown that loss of water molecules upon dehydration could actually increase the number of hydrogen bonds between residues of the protein, hence increasing the stability of the protein in the gas phase (van der Spoel et al., 2011van der Spoel D. Marklund E.G. Larsson D.S. Caleman C. Proteins, lipids, and water in the gas phase.Macromol. Biosci. 2011; 11: 50-59Crossref PubMed Scopus (68) Google Scholar). Our understanding of the mechanisms enabling protein complexes to be retained close to their native state following loss of water have been strengthened by 20 years of research. Recent experimental insights come from electron capture dissociation (ECD), in which folded structure is more resistant to backbone fragmentation (Skinner et al., 2013Skinner O.S. Breuker K. McLafferty F.W. Charge site mass spectra: conformation-sensitive components of the electron capture dissociation spectrum of a protein.J. Am. Soc. Mass Spectrom. 2013; 24: 807-810Crossref PubMed Scopus (24) Google Scholar), and ion mobility (IM) MS (Wyttenbach and Bowers, 2011Wyttenbach T. Bowers M.T. Structural stability from solution to the gas phase: native solution structure of ubiquitin survives analysis in a solvent-free ion mobility-mass spectrometry environment.J. Phys. Chem. B. 2011; 115: 12266-12275Crossref PubMed Scopus (257) Google Scholar), a technique that measures the orientationally averaged collision cross-section (CCS) of a protein or its complex. Both of these methods have shown that compact native-like structures can survive in the gas phase of the mass spectrometer, at least on the millisecond timescale, providing that the electrospray conditions are controlled so that desolvation is achieved with minimal activation. The ability to measure CCS and correlate these experimental values with theoretical cross-sections calculated from X-ray structures has allowed quantitative comparison to the structures observed in the gas phase (Bush et al., 2010Bush M.F. Hall Z. Giles K. Hoyes J. Robinson C.V. Ruotolo B.T. Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology.Anal. Chem. 2010; 82: 9557-9565Crossref PubMed Scopus (606) Google Scholar). While some structural collapse is evident both in MD simulations and in the CCS measured experimentally (Hall et al., 2012Hall Z. Politis A. Robinson C.V. Structural modeling of heteromeric protein complexes from disassembly pathways and ion mobility-mass spectrometry.Structure. 2012; 20: 1596-1609Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), much progress has been made in relating these experimental CCSs to structure. This has led to the use of CCS restraints to guide structural modeling of the overall topologies of unknown complexes (Leary et al., 2009Leary J.A. Schenauer M.R. Stefanescu R. Andaya A. Ruotolo B.T. Robinson C.V. Thalassinos K. Scrivens J.H. Sokabe M. Hershey J.W. Methodology for measuring conformation of solvent-disrupted protein subunits using T-WAVE ion mobility MS: an investigation into eukaryotic initiation factors.J. Am. Soc. Mass Spectrom. 2009; 20: 1699-1706Crossref PubMed Scopus (49) Google Scholar, Politis et al., 2013Politis A. Park A.Y. Hall Z. Ruotolo B.T. Robinson C.V. Integrative modelling coupled with ion mobility mass spectrometry reveals structural features of the clamp loader in complex with single-stranded DNA binding protein.J. Mol. Biol. 2013; PubMed Google Scholar, Pukala et al., 2009Pukala T.L. Ruotolo B.T. Zhou M. Politis A. Stefanescu R. Leary J.A. Robinson C.V. Subunit architecture of multiprotein assemblies determined using restraints from gas-phase measurements.Structure. 2009; 17: 1235-1243Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Deciphering the subunit composition of protein-protein complexes using MS has long been the hallmark of the MS of soluble protein complexes. Recently this enabled description of a multipart reaction cycle involving complexes formed between Hsp90, Hop, Hsp70, and FKBP52. By defining a series of KD values, it was possible to deduce complexes likely to be populated within the cell (Ebong et al., 2011Ebong I.O. Morgner N. Zhou M. Saraiva M.A. Daturpalli S. Jackson S.E. Robinson C.V. Heterogeneity and dynamics in the assembly of the heat shock protein 90 chaperone complexes.Proc. Natl. Acad. Sci. USA. 2011; 108: 17939-17944Crossref PubMed Scopus (59) Google Scholar). Protein-protein interactions within Mega Dalton virus capsids have also been maintained in the gas phase, revealing the nature of the interactions between the viral genome and capsid proteins and prompting a proposal for the mechanism of the alkaline-triggered uncoating reaction (Snijder et al., 2013Snijder J. Uetrecht C. Rose R.J. Sanchez-Eugenia R. Marti G.A. Agirre J. Guérin D.M. Wuite G.J. Heck A.J. Roos W.H. Probing the biophysical interplay between a viral genome and its capsid.Nat. Chem. 2013; 5: 502-509Crossref PubMed Scopus (102) Google Scholar). As the complexity of soluble proteins studied by MS continues to grow, this has broadened its application to an ever increasing series of challenging biomolecules, including those involved in amyloidosis (Ashcroft, 2010Ashcroft A.E. Mass spectrometry and the amyloid problem—how far can we go in the gas phase?.J. Am. Soc. Mass Spectrom. 2010; 21: 1087-1096Crossref PubMed Scopus (56) Google Scholar) and in interactions with nucleic acids (Park and Robinson, 2011Park A.Y. Robinson C.V. Protein-nucleic acid complexes and the role of mass spectrometry in their structure determination.Crit. Rev. Biochem. Mol. Biol. 2011; 46: 152-164Crossref PubMed Scopus (13) Google Scholar). One class of protein complexes that had eluded MS for more than a decade was membrane protein complexes, the main focus of this review. We will trace the history of the development of MS for these complexes. In earlier papers that questioned the folded state of soluble proteins in vacuo, membrane proteins were predicted to retain their native fold (Wolynes, 1995Wolynes P.G. Biomolecular folding in vacuo!!!(?).Proc. Natl. Acad. Sci. USA. 1995; 92: 2426-2427Crossref PubMed Scopus (119) Google Scholar). The low dielectric interior of lipid bilayers was suggested to mimic closely the vacuum conditions of a mass spectrometer (Jarrold, 2007Jarrold M.F. Helices and sheets in vacuo.Phys. Chem. Chem. Phys. 2007; 9: 1659-1671Crossref PubMed Scopus (111) Google Scholar). Given these favorable predictions for membrane proteins, much effort ensued to deliver membrane complexes from solution to the gas phase (Ilag et al., 2004Ilag L.L. Ubarretxena-Belandia I. Tate C.G. Robinson C.V. Drug binding revealed by tandem mass spectrometry of a protein-micelle complex.J. Am. Chem. Soc. 2004; 126: 14362-14363Crossref PubMed Scopus (60) Google Scholar, Lengqvist et al., 2004Lengqvist J. Svensson R. Evergren E. Morgenstern R. Griffiths W.J. Observation of an intact noncovalent homotrimer of detergent-solubilized rat microsomal glutathione transferase-1 by electrospray mass spectrometry.J. Biol. Chem. 2004; 279: 13311-13316Crossref PubMed Scopus (42) Google Scholar). While these early attempts showed great promise in delivering integral membrane proteins, it was not clear whether specific interactions could be maintained. A breakthrough in the field came however with the first demonstration that transmembrane and cytoplasmic subunits could be maintained intact in the gas phase of the mass spectrometer in a heterotetramer (Barrera et al., 2008Barrera N.P. Di Bartolo N. Booth P.J. Robinson C.V. Micelles protect membrane complexes from solution to vacuum.Science. 2008; 321: 243-246Crossref PubMed Scopus (282) Google Scholar; Figure 1). This was therefore an important phenomenological advance because it implied that once released from detergent micelles, the hydrophobic environment of the gas phase was sufficient to maintain interactions in heteromeric membrane protein complexes. Analogous to the situation for soluble complexes, MS is of particular interest when applied to the study of membrane complexes of unknown stoichiometry. The fact that this information is often challenging to obtain from traditional approaches, due to the presence of the micelle surrounding the membrane protein changing its biophysical properties, means that MS is emerging as a powerful tool for the determination of membrane subunit stoichiometry. Classical methods such as multi angle laser light scattering (MALLS; Oliva et al., 2004Oliva A. Llabrés M. Fariña J.B. Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins.Curr. Drug Discov. Technol. 2004; 1: 229-242Crossref PubMed Scopus (26) Google Scholar), analytical ultra-centrifugation (AUC; Howlett et al., 2006Howlett G.J. Minton A.P. Rivas G. Analytical ultracentrifugation for the study of protein association and assembly.Curr. Opin. Chem. Biol. 2006; 10: 430-436Crossref PubMed Scopus (148) Google Scholar), and native gel or size exclusion chromatography (SEC; Hong et al., 2012Hong P. Koza S. Bouvier E.S. Size-exclusion chromatography for the analysis of protein biotherapeutics and their aggregates.J. Liquid Chromatogr. Relat. Technol. 2012; 35: 2923-2950PubMed Google Scholar) can be used to gather information concerning the size of a membrane protein. However, these techniques are of poor accuracy or have inherent problems due to the presence of the detergent. Both MALLS and SEC provide information on the entire protein micelle complex rather than the protein alone. For AUC, the presence of the micelle affects buoyancy making the protein "float" and reducing its sedimentation coefficient. Determination of the molecular mass of a membrane protein with AUC thus requires the prior quantification of detergent bound to the membrane protein, typically using radiolabeled detergent, and a combination of SEC to get the hydrodynamic radius and sedimentation velocity or equilibrium analysis to obtain the sedimentation coefficient (le Maire et al., 2008le Maire M. Arnou B. Olesen C. Georgin D. Ebel C. Møller J.V. Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca2+-ATPase as an example.Nat. Protoc. 2008; 3: 1782-1795Crossref PubMed Scopus (68) Google Scholar). Alternatively, the micelle can be circumvented using deuterated detergents and performing small angle neutron scattering (SANS; Wang et al., 2003Wang Z.Y. Muraoka Y. Nagao M. Shibayama M. Kobayashi M. Nozawa T. Determination of the B820 subunit size of a bacterial core light-harvesting complex by small-angle neutron scattering.Biochemistry. 2003; 42: 11555-11560Crossref PubMed Scopus (18) Google Scholar). A recent method has also been proposed that requires the insertion of the protein of interest into nanodiscs and uses a combination of dynamic light scattering and AUC (Inagaki et al., 2013Inagaki S. Ghirlando R. Grisshammer R. Biophysical characterization of membrane proteins in nanodiscs.Methods. 2013; 59: 287-300Crossref PubMed Scopus (78) Google Scholar). In this context, the use of MS to assess the molecular mass and subunit stoichiometry of intact membrane complexes represents a significant advance because it allows accurate mass measurement in the absence of detergent. The success and potential interest of using MS approaches to study intact membrane protein assemblies prompted a better understanding of the fundamental mechanisms by which micelles are removed in the gas phase. The high collision energy required to release membrane proteins from micelles implies that most of this energy is dissipated within the dissociating detergent molecules. This would protect the membrane protein complexes from unfolding or dissociation of protein subunits, as originally suggested (Barrera et al., 2008Barrera N.P. Di Bartolo N. Booth P.J. Robinson C.V. Micelles protect membrane complexes from solution to vacuum.Science. 2008; 321: 243-246Crossref PubMed Scopus (282) Google Scholar). Recent evidence implies that the energy required to release membrane proteins from micelles depends both on the detergent and protein or complex. We will consider the process for empty detergent micelles first and then those containing a protein. Considering first the dissociation process of empty detergent micelles in the gas phase, in the absence of proteins, sizes of gas phase detergent clusters were compared with solution phase aggregation numbers. Results clearly showed that the size and distribution of nC-trimethyl ammonium bromide (ncTAB) micelles formed in solution were not maintained in the gas phase (Borysik and Robinson, 2012aBorysik A.J. Robinson C.V. Formation and dissociation processes of gas-phase detergent micelles.Langmuir. 2012; 28: 7160-7167Crossref PubMed Scopus (28) Google Scholar), compromising attempts to determine critical micellar concentrations using MS. Evaporation of neutral detergent molecules appears to be a critical force in driving micelle dissociation (Borysik and Robinson, 2012bBorysik A.J. Robinson C.V. The 'sticky business' of cleaning gas-phase membrane proteins: a detergent oriented perspective.Phys. Chem. Chem. Phys. 2012; 14: 14439-14449Crossref PubMed Scopus (31) Google Scholar). The small aquaporin Pagp from Escherichia coli was selected as a model system to study its mechanism of release from micelles in the gas phase. Following detachment from dodecyl maltoside (DDM) micelles, subsequent IM-MS revealed that this integral β-barrel protein exists in two conformations in the gas phase: one corresponding to a native-like structure and the other showing partial collapse of the external loops (Borysik et al., 2013Borysik A.J. Hewitt D.J. Robinson C.V. Detergent release prolongs the lifetime of native-like membrane protein conformations in the gas-phase.J. Am. Chem. Soc. 2013; 135: 6078-6083Crossref PubMed Scopus (49) Google Scholar). Given the low activation conditions required to release the protein from the micelle, differing numbers of DDM molecules (from 1 to 6) remained attached to the protein. Surprisingly, the number of bound detergent molecules was found to be inversely proportional to the population of native-like protein remaining. This observation implies therefore that the protein is not protected by the detergent per se but rather by its release, suggesting a mechanism akin to evaporative cooling, which is proposed more generally for electrospray ions with solution additives (Bagal et al., 2009Bagal D. Kitova E.N. Liu L. El-Hawiet A. Schnier P.D. Klassen J.S. Gas phase stabilization of noncovalent protein complexes formed by electrospray ionization.Anal. Chem. 2009; 81: 7801-7806Crossref PubMed Scopus (58) Google Scholar). Our current understanding of the release of protein complexes from micelles in the gas phase is that internal energy of the protein in a micelle has to be increased to a level that is lower than that at the onset of unfolding (Figure 2). Part of this energy can be dissipated via neutral loss of detergent molecules. The amount of energy transferred to these detergent molecules depends on the nature of the detergent, the interface with the protein and the size of the cluster. This energy component should then determine the proportion of native-like membrane protein or complexes maintained in the gas phase. This first mass spectra of an intact membrane protein complex (BtuC2D2) with transmembrane and cytoplasmic subunits remaining associated was swiftly followed by reports of five multidrug transporters (Barrera et al., 2009Barrera N.P. Isaacson S.C. Zhou M. Bavro V.N. Welch A. Schaedler T.A. Seeger M.A. Miguel R.N. Korkhov V.M. van Veen H.W. et al.Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions.Nat. Methods. 2009; 6: 585-587Crossref PubMed Scopus (143) Google Scholar, Lin et al., 2009Lin H.T. Bavro V.N. Barrera N.P. Frankish H.M. Velamakanni S. van Veen H.W. Robinson C.V. Borges-Walmsley M.I. Walmsley A.R. MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.J. Biol. Chem. 2009; 284: 1145-1154Crossref PubMed Scopus (76) Google Scholar, Velamakanni et al., 2009Velamakanni S. Lau C.H. Gutmann D.A. Venter H. Barrera N.P. Seeger M.A. Woebking B. Matak-Vinkovic D. Balakrishnan L. Yao Y. et al.A multidrug ABC transporter with a taste for salt.PLoS ONE. 2009; 4: e6137Crossref PubMed Scopus (32) Google Scholar; Figure 1). Of particular interest in these studies was the observation of lipids that appeared to be integrated within the structure. The early studies assigned lipids as being bound at the interface between monomers because lipid binding was not observed for dissociated subunits. Taking into account the mass associated with bound lipids turned out to be one of the defining moments in understanding the spectra of the rotary ATPases (Zhou et al., 2011Zhou M. Morgner N. Barrera N.P. Politis A. Isaacson S.C. Matak-Vinković D. Murata T. Bernal R.A. Stock D. Robinson C.V. Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding.Science. 2011; 334: 380-385Crossref PubMed Scopus (219) Google Scholar). Recording the mass spectra of rotary ATPases, while undoubtedly challenging, was not the limiting step; assigning the spectra with unknown stoichiometries of protein subunits, lipids, and nucleotides, associated with the various subcomplexes, was not possible using the protocols available at that time. This prompted development of new software to simulate and assign mass spectra from complex heterogeneous assemblies (Morgner and Robinson, 2012Morgner N. Robinson C.V. Massign: an assignment strategy for maximizing information from the mass spectra of heterogeneous protein assemblies.Anal. Chem. 2012; 84: 2939-2948Crossref PubMed Scopus (114) Google Scholar). Comparison of two V-type ATPases from Thermus thermophilus and Enterococcus hirae clearly showed different lipid binding patterns in the membrane rings. This highlights the ability of the complexes to tailor the lipid plug to adapt the central orifice of the membrane ring to fit the central rotor. Interestingly lipid specificity seems to be driven by the size of both the ring and of the central stalk with which it interacts. A larger reduction of the membrane ring orifice was observed for the F-type ATPase from S. oleracea (10–20 Å final; Sch
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