- Email: [email protected]
Protein folding and interactions revealed by mass spectrometry
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Protein folding and interactions revealed by mass spectrometry Alexander M Last and Carol V Robinson* Mass spectrometry is capable of examining very large, dynamic proteins and this ability, coupled with its relatively high throughput and low sample requirements, is reflected by its increasing importance for the characterisation of protein structure. Recent developments in mass spectrometry, in particular the refinement of the electrospray process and its coupling with time-of-flight mass analysis, mean that it is poised to contribute not only as a complementary tool but also with a defined role in many areas of chemical biology.
study of noncovalent interactions by MS. The current view is that many more conformations are available to gas-phase ions [6••], depending on the time scale and conditions of the experiment, but that some elements of secondary structure are retained. The overwhelming number of examples, however, where both stoichiometry and even relative affinity of binding diverse ligands to protein targets, provides compelling evidence for preservation of some tertiary structure under carefully controlled conditions.
Addresses Oxford Centre for Molecular Sciences, South Parks Road, Oxford OX1 3QT, UK *e-mail: [email protected]
Initial investigations of protein–ligand complexes using MS indicated an enhanced sensitivity for ionic interactions over hydrophobic ones [7,8]. Numerous examples have been reported of ionic interactions involving metals binding to proteins, including metallothioneins that form intermediates with 4Cu(I) ion clusters before the native 6Cu(I) species [9], and zinc ions bound to hexameric insulin complexes [10]. The refinement of the electrospray process, with the introduction of the nanoflow technique, in which both the size of the needle orifice and the flow rate of the sample are reduced, has led to a smaller droplet size requiring much milder desolvation. This, coupled with the lower voltages in the source, has enabled the survival of weakly associated protein–ligand complexes and allowed the contribution of hydrophobically driven interactions to be addressed [11••]. For example, MS analysis of the binding of aldose reductase to four different inhibitors showed that the strength of binding in the gas phase could be correlated to the electrostatic and hydrogen-bonding interactions defined by X-ray analysis, assuming that the hydrophobic component to the binding energy remained constant [12•].
Current Opinion in Chemical Biology 1999, 3:564–570 1367-5931/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviations FHV Flock House virus FTICR Fourier-transform ion cyclotron resonance HX hydrogen exchange MS mass spectrometry RBP retinol-binding protein TTR transthyretin VLP virus-like particles
Introduction The major incentive behind the design and implementation of new technologies in mass spectrometry (MS) is the drive to obtain increased resolution and mass accuracy, together with an increase in the efficiency of the ionisation processes. In pursuit of these objectives, the introduction of nanoflow electrospray [1] and its marriage with time-offlight [2], and its increased efficiency with Fourier-transform ion cyclotron resonance (FTICR) MS [3], have had a major impact. The introduction of novel combinations such as quadrupole time-of-flight [4] has also extended the tandem capabilities of MS. Our focus in this review is to bring together a diverse range of applications involving electrospray that have arisen largely from these developments. We also highlight the level of structural information available from both individual proteins and their complexes.
Protein–ligand interactions The technique of electrospray produces a stream of highly charged droplets by the continuous flow of a protein solution through a needle maintained at high voltage [5]. A series of evaporation and desorption events produce gas phase ions that are subsequently attracted into the mass spectrometer by electric and pressure gradients. The extent to which the conformation of the gas phase ion produced as a result of this process mirrors that of the protein in solution is an area of major research, not only because of its scientific interest but also because of its impact on the
The ionic interactions between proteins and nucleic acids and the effect of cofactors are readily observed. For example, the trp repressor homodimer binds to a 21-base-pair DNA sequence, yet remains monomeric in the absence of this sequence [13] while the zinc-mediated binding of the nucleocapsid protein (NCp7) to NCp7-psi RNA was not observed in the absence of the metal ion [14]. Metal cofactors have also been investigated in the context of the vitamin D receptor binding to double-stranded DNA [15]. The bacteriophage T4 regA protein in the presence of target sequences of RNA showed stable gas-phase complexes that were resistant to dissociation in the gas phase [16]. Fragmentation of the peptide backbone occurred before the complex was dissociated, providing insight into the RNA-binding site. An alternative MS-based method for identifying DNA-binding sites in a protein has been described and involves chemical derivatisation of the lysine sidechains involved in a protein–DNA interaction [17•]. Small peptide ligands, such as those derived from the bacterial cell wall, and their interactions with the glycopeptide
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Figure 1 Nanoflow electrospray ionisation mass spectra showing the effect of increasing collision energy on the complex formed between the Fyn SH2 domain and the peptide EPQY*EEEPIYL (single letter amino acid code, where Y* is phosphotyrosine). The charge states of each mass spectrum have been converted onto a mass scale and are normalised according to the actual intensities of the spectra. At low cone voltage (35 V) the water-free protein–peptide complex is the dominant species (peak A). Increasing the cone voltage to 80 V disrupts the complex and the apo-protein now predominates (peak B). Further increases in the collision energy (to a cone voltage of 100 V and 140 V) leads to a further disruption of the complex. The water-bound complex, however, is now more intense than the water-free complex (peaks C and D). Dashed lines are used to align the corresponding peaks in each spectrum.
apo-SH2 SH2–EPQY*EEEPIYL 100
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antibiotics vancomycin and ristocetin, have been examined by MS and the relative binding affinities assessed [18]. In addition, peptides from HIV-1 were bound to a 17-amino-acid microantibody and the complex was examined using MS; key residues in the HIV peptides were revealed by enzymatic digestion [19 •] — binding decreased dramatically as certain residues were removed, thus revealing the residues required for high-affinity interactions. The binding of peptide mimics of protein ligands to the Src homology 2 (SH2) domain from Src tyrosine kinases was examined by MS. Analysis of the spectra revealed peaks with masses that correspond to the presence of water molecules attached to the apo-protein and peptide-bound complex [20]. The numbers of water molecules bound to the complex in the gas phase correlates well with the tightly bound molecules seen in the crystal structure. In addition, recent evidence has shown that for the SH2 domain from Fyn, three water molecules remain tightly bound even after the energy of collisions is increased. This observation strongly suggests that these water molecules mediate interactions in the ligand-binding interface, conferring an additional stability to the complex even in the gas phase [11••] (Figure 1).
Counting individual atoms An electrospray mass spectrum comprises a series of peaks, known as a ‘charge state series’, from which the mass of a protein can be determined. The width of a charge state arises from the convolution of naturally occurring isotopes within the protein, principally the 13C atoms, and the resolving power of the instrument. By using proteins that have minimal amounts of 13C and 15N, the width of the peak is greatly reduced [21]. Using FTICR, ultra-high resolution was obtained, enabling peaks arising from isotopic fine structure to be separated. For example, using a doubly depleted 13C and 15N sample of the tumour suppressor
13,000 Mass
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protein p16, it was possible to determine the number of sulfur atoms directly from the ultra-high-resolution mass spectrum as 5 ± 0.3 without use of any other information about the protein (Figure 2) [22]. Counting atoms forms the basis of measuring the conformational dynamics of proteins by hydrogen exchange (HX). In this process, a fully protonated protein is diluted Figure 2
∗
∗
# [M+16H]16+
Monoisotopic peak
988.0
988.5
#18O
#34S
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m/z FTICR mass spectrum of p16 tumor suppressor protein showing the monoisotopic peak for the protein and the peaks assigned to the protein containing 34S and 18O atoms. The isotope distribution of the +16 charge state is inset, and an expansion of the labelled peaks is shown. The asterisk represents the peak resulting from the monoisotopic mass and the hash represents the peak resulting from addition of the two Daltons, assigned to a species containing either a single atom of 34S or 18O. From the ratio of the heights of the monoisotopic and the 34S peaks, the number of sulfur atoms in the protein was determined as 5 ± 0.3. Reproduced with permission from Proc Nat Acad Sci USA. m/z, mass/change ratio.
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into a deuterated buffer. All labile protons in sidechains and solvent-exposed backbone amide protons will exchange and the number of deuterons incorporated provides a measure of the conformational dynamics or stability of the protein [23]. When HX is followed by enzymatic digestion and high-performance liquid chromatography (HPLC) separation, it is possible to locate regions of the protein that are protected against exchange with solvent. Using this approach with FTICR analysis, several regions of the variant form of Yersinia protein tyrosine phosphatase were found to be more dynamic than those of its wild type counterpart, and binding of an inhibitor was found to decrease the flexibility of the wild type protein but increase that of the mutant [24••]. This approach has also shown that two types of protein domains, the SH2 and SH3 domains from Hck, have different HX kinetics whether expressed in isolation or as a single polypeptide chain [25•]. Thus the dynamics and solvent accessibility of regions of each domain are altered in the two domain construct, changing the rate at which protons are able to exchange for solvent deuterons. Using a combined NMR and MS approach, cooperative HX in the third domain of the turkey protein ovomucoid showed that the number of amide protons exchanging in a cooperative manner is lower than that predicted by NMR measurements alone [26•].
Site specificity in hydrogen exchange labelling Locating the individual backbone amides that are protected from exchange can be achieved through two distinct methods. The first, mentioned above, employs a combination of enzymatic digestion and HPLC separation of the resulting fragments in which deuterium labelling is preserved. Alternatively, it has been proposed that collision-induced dissociation under high-energy conditions may yield peptide fragments from which HX protection can be deduced. Initial investigations of cytochrome c showed that under the high-energy collision-induced dissociation conditions, deuterons had migrated along the backbone of the protein leading to scrambling of the labelling information [6••]. Interestingly, these observations led to the proposal that under electron capture conditions collision-induced migration of deuterons may be significantly reduced [27,28••]. Moreover, it has recently been demonstrated that under some circumstances it may be possible to use collisioninduced dissociation of peptides to locate deuterons [29••]. HX of native cytochrome c followed by peptic digestion and MS analysis, showed that amino-terminally derived fragments, formed by cleavage across amide linkages, gave rise to retention of deuterium label, while carboxy-terminal fragments lead to hydrogen rearrangements. The apparent conflict between the results from peptides derived from enzymatic digestion and the intact protein presumably arises from the lower enery required to fragment the former.
Observation of folding intermediates The ability of MS to distinguish between populations of molecules with different amounts of deuterium label
enables the study of the heterogeneity of protein folding reactions. Using a pulse labelling method, partially structured species have been identified that incorporate intermediate numbers of deuterons during the folding reaction (i.e. between the number in the fully unfolded and in the native protein). Previous studies of hen egg white lysozyme, a two domain protein with one region having mainly α-helical structure (the α-domain) and the other consisting predominantly of β-sheet (the β-domain), have shown multiple folding pathways when refolding is carried out at pH 5.0. Approximately 30% of molecules are rapidly protected from HX in both domains. The remainder proceeds via a well-defined intermediate corresponding in mass to persistent structure in the α-domains. At high temperature, MS has shown the loss of the α-domain intermediate, whereas high salt concentrations were found to regenerate the properties associated with this intermediate [30]. The effect of metal ions on refolding was found to retard a late step in folding, possibly the docking of two pre-formed domains, whereas refolding at nearer physiological pH shows that all molecules fold via an obligatory intermediate [31]. The interactions of lysozyme folding intermediates with the molecular chaperone GroEL showed that the chaperone interacts with a species late in the folding reaction that was already sufficiently compact to have native-state HX protection [32]. The oxidative refolding of hen lysozyme analysed by enzymatic digestion, FTICR and tandem MS [33•] showed that the last disulfide bond to form was the one linking the two domains, which is consistent with the nonoxidative refolding, where the final docking constitutes a late folding event. Using a combined HX labelling MS approach, the folding pathway of apomyoglobin was also shown to proceed via a discrete intermediate [34]. However, the refolding of the calcium-binding protein bovine α-lactalbumin, which is structurally homologous to the C-type lysozymes, showed no well-defined intermediates by MS. Rather, in its apoform the protein was found to undergo rapid collapse to a molten globule-type intermediate, followed by a slow rearrangement to the native state [35]. The addition of Ca2+ ions to the refolding buffer was found to accelerate this rearrangement to the native state by a factor of two orders of magnitude. HX labelling has also been employed to study the refolding of two variants of human lysozyme that form amyloid fibrils in vivo. Human lysozyme has two naturally occurring variants, Ile56→Thr and Asp67→His, that have been shown, using HX and MS, to have enhanced conformational dynamics relative to their wild type counterpart [36]. In additional experiments, the rates of formation of folding intermediates indicate that the Ile56→Thr variant, which disrupts the interface between the two domains of the protein, decelerates the conversion from a late intermediate to the native state [37 •] (Figure 3). In contrast, the Asp67→His variant has no observable effect on the formation of the native state.
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Protein association Wild type and variant forms of human transthyretin (TTR) can also form amyloid fibrils in vivo. The onset of clinical symptoms (i.e. familial amyloid neuropathy) resulting from amyloid deposition occurs earlier if a variant is present, suggesting that mutation has decreased the stability of the protein. TTR is functional as a homotetramer and by comparing the severity of MS conditions required to disrupt this tetramer it was found that the wild type protein was much more resistant to collision-induced dissociation than either the Leu55→Pro or Val30→Met variants [38•] (Figure 4). Lowering the ionic strength results in a less stable tetramer, whereas addition of thyroxine, the hormone carried by TTR, increases the stability of the intact species.
Figure 3
IIe56-Thr
Wild type
N U
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I
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The DNA-binding protein HU is functional as a dimer but remains in a monomeric, unfolded state if present in solution at low salt concentration [39••]. As the ionic strength is increased, the monomer becomes more compact, reflected in the decreased numbers of charges per monomer, and then assembles to the dimer. The HX rates of this protein decrease as ionic strength rises (i.e. as the proportion of the unfolded monomer decreases). In a separate investigation, two structurally similar forms of HU, derived from related bacteria, were able to form a heterodimer when a mixture of the unfolded proteins was maintained at low salt concentration or high temperature. Under these conditions, the unfolded proteins were able to refold and calculation of the binding energies of the homodimers was possible [40].
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Electrospray mass spectra of the refolding of the Ile56→Thr variant and 15N-labelled wild type human lysozymes from 0–2 s. The peaks are labelled U, I and N and represent unfolded, intermediate and native-state populations, respectively. m/z, mass to charge ratio.
Figure 4
(a) Electrospray mass spectra of the wild type TTR tetramer at different cone voltages. At low cone voltages, the predominant species is that of the tetramer (between mass/charge [m/z] ratios of 3,000–4,000), whereas at high cone voltage, only the monomeric species is detected. The mass/charge ratio of the tetramer is markedly different from the monomer due to formation of salt bridges in the intact species reducing the number of ionizable groups, and the
presence of counter-ions in the macromolecular structure. The asterisks denote fragmentation of the peptide backbone at proline 11. (b) The effects of increasing cone voltage on the proportions of tetramer present in the mass spectrum as a percentage of total TTR for wild-type (circle), Val30→Met (square) and Leu55→Pro (diamond). Note that the tetramers formed for both variants are markedly less stable than that formed with the wild-type protein.
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Subunit packing in macromolecular complexes One of the most exciting applications of MS has been to show that crystallographically identical virus structures do not necessarily share identical dynamics [41••]. Flock House virus (FHV) reproduction is altered depending on the host organism. In Drosophila, the correct viral RNA is packaged, forming an FHV particle; in baculovirus, however, virus-like particles (VLPs), containing cellular RNA, are formed. X-ray crystallographic analyses of FHV and VLPs show that the capsid structures are identical. When the FHV and VLPs are exposed to enzymatic digestion and covalent modification, however, the rates of reaction were found to be faster for the VLPs than for the FHV, suggesting that the viral RNA contributes to the overall stability of the capsid structure. Similarly, the VP4 coat protein of human rhinovirus 14 is located beneath the surface of the capsid, yet is readily digested by trypsin [42••]. This result is interpreted in terms of ‘breathing’ of the capsid, thus allowing trypsin to permeate and interact with a protein not thought to be exposed to solvent. Addition of an antiviral drug reduces the amount of digestion products formed and hence reduces the fluctuations of the capsid surface. As well as being the thyroxine carrier in blood, TTR also forms a physiologically important complex with retinolbinding protein (RBP). Several TTR–RBP complexes, with differing stoichiometries, are feasible. Simulation of the overlapping charge states allows determination of the relative amounts of the different complexes with one or two RBP molecules bound. From this simulation, dissociation constants were calculated and the results show that binding of the first RBP has negative cooperativity for the binding of the second [43]. Moreover, dissociation in the gas phase was used to identify the connectivities between the different subunits. This approach has also been employed in the study of ribosomes from Escherichia coli. Despite the size and complexity of the 2.3 MDa particle, well-resolved spectra of the dissociation products have been obtained, enabling the topology of the particle to be examined [44••]. A dramatic example of both the maintenance and disassembly of macromolecular complexes is that of an 800 kDa complex of the molecular chaperone GroEL from E. coli [45••,46]. Well-defined charge states for the intact complex were observed at a mass/charge value of 10,000, from which the mass was calculated as 803,742 ± 616 Da. Collisioninduced dissociation of this macromolecular complex showed that the symmetry of the subunit packing in the two heptameric rings was retained even in the gas phase.
Conclusions The phenomenal growth of mass spectrometry in recent years is due not only to its capabilities in defining the primary structure of proteins from electrophoretic gels, but also because of its applications to noncovalent interactions and folding, as described herein. The numerous examples where both stoichiometry and relative affinity in the bind-
ing of protein ligand and macromolecular complexes correlates with solution conditions puts the methodology beyond reasonable doubt. As MS enters a new era — studying the assembly of macromolecular complexes — the many and varied investigations of protein conformation and interactions lay the foundation for the use of MS in the wider context of structural biology.
Acknowledgements We acknowledge, with thanks, many helpful discussions with the Robinson MS group and with Chris Dobson. This contribution was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), Engineering and Physical Science Research Council (EPSRC), and the Medical Research Council. Carol Robinson also acknowledges support from the Royal Society.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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McLafferty FW, Guan ZQ, Haupts U, Wood TD, Kelleher NL: Gaseous conformational structures of cytochrome c. J Am Chem Soc 1998, 120:4732-4740. The location of deuterium label in cytochrome c was examined by collisioninduced dissociation and, despite extensive scrambling of labile deuterons, the results show that four small helical regions are maintained, even in the most open conformations. 7.
Robinson CV, Chung EW, Kragelund BB, Knudsen J, Aplin RT, Poulsen FM, Dobson CM: Probing the nature of non-covalent interactions by mass spectrometry. A study of protein-CoA ligand binding and assembly. J Am Chem Soc 1996, 118:8646-8653.
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Wu QY, Gao JM, Joseph-McCarthy D, Sigal GB, Bruce JE, Whitesides GM, Smith RD: Carbonic anhydrase-inhibitor binding: from solution to the gas phase. J Am Chem Soc 1997, 119:1157-1158.
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Jensen LT, Peltier JM, Winge DR: Identification of a four copper folding intermediate in mammalian copper metallothionein by electrospray ionization mass spectrometry. J Biol Inorg Chem 1998, 3:627-631.
10. Fabris D, Fenselau C: Characterisation of allosteric insulin hexamers by electrospray ionization mass spectrometry. Anal Chem 1999, 71:384-387. 11. Chung EW, Henriques D, Renzoni D, Ladbury JE, Robinson CV: •• Probing the nature of interactions in SH2 binding interfaces — evidence from electrospray mass spectrometry. Protein Sci 1999, in press. This paper shows that under high-energy collision conditions up to three water molecules remain bound to the complex, presumably as a result of hydrogen bonding interactions in the ligand binding interface. 12. Rogniaux H, Van Dorsselaer A, Barth P, Biellmann JF, Barbanton J, • van Zandt M, Chevrier B, Howard E, Mitschler A, Potier N et al.: Binding of aldose reductase inhibitors: correlation of
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crystallographic and mass spectrometric studies. J Am Soc Mass Spectrom 1999, 10:635-647. In this study, both X-ray crystallography and mass spectrometry were applied to aldose reductase and four potential inhibitors and the results were used to demonstrate the complementarity between the two techniques. 13. Potier N, Donald LJ, Chernushevich I, Ayed A, Ens W, Arrowsmith CH, Standing KG, Duckworth HW: Study of a noncovalent trp repressor:DNA operator complex by electrospray time-of-flight mass spectrometry. Protein Sci 1998, 7:1388-1395. 14. Loo JA, Holler TP, Foltin SK, McConnell P, Banotai CA, Horne NM, Mueller WT, Stevenson TI, Mack DP: Application of electrospray ionization mass spectrometry for studying human immunodefeciency virus protein complexes. Proteins 1998, 2(suppl):28-37. 15. Veenstra TD, Benson LM, Craig TA, Tomlinson AJ, Kumar R, Naylor S: Metal mediated sterol receptor–DNA complex association and dissociation by electrospray ionization mass spectrometry. Nat Biotechnol 1998, 16:262-266. 16. Liu CL, Tolic LP, Hofstadler SA, Harms AC, Smith RD, Kang CH, Sinha N: Probing regA/RNA interactions using electrospray ionization Fourier transform ion cyclotron resonance-mass spectrometry. Anal Biochem 1998, 262:67-76. 17. •
Chen JW, Smith DL, Griep MA: The role of the 6 lysines and the terminal amine of Escherichia coli single-strand binding protein in its binding of single-stranded DNA. Protein Sci 1998, 7:1781-1788. The authors present a method for covalent modification of lysine residues allowing differential labelling to probe the accessibility of the sidechain amino group and their interactions with DNA. 18. Jorgensen TJD, Roepstorff P, Heck AJR: Direct determination of solution binding constants for noncovalent complexes between bacterial cell wall peptide analogues and vancomycin group antibiotics by electrospray ionization mass spectrometry. Anal Chem 1998, 70:4427-4432.
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26. Arrington CB, Teesch LM, Robertson AD: Defining protein • ensembles with native state NH exchange: kinetics of interconversion and cooperative units from combined NMR and MS analysis. J Mol Biol 1999, 285:1265-1275. NMR-derived HX data of the turkey ovomucoid domain are used to simulate peak widths during mass spectrometry (MS) exchange. Observed MS peaks do not match these, suggesting that the extent of cooperativity is more limited than suggested using NMR. 27.
Zubarev RA, Kelleher Nl, McLafferty FW: Electron capture dissociation of multiply charged protein cations. A nonergodic process. J Am Chem Soc 1998, 120:3265-3266.
28. Zubarev RA, Kruger NA, Frediksson EK, Lewis MA, Horn DM, •• Carpenter BK, McLafferty FW: Electron capture dissociation of gaseous multiply charged proteins is favoured at disulphide bonds and other sites of high hydrogen atom affinity. J Am Chem Soc 1999, 121:2857-2862. The electron capture dissociation method was shown to generate preferential cleavage of the backbone at disulfide bonds, in contrast to excitation of the ions by photons or low-energy collisions. 29. Deng YZ, Pan H, Smith DL: Selective isotope labeling •• demonstrates that hydrogen exchange at individual peptide amide linkages can be determined by collision-induced dissociation mass spectrometry. J Am Chem Soc 1999, 121:1966-1967. Hydrogen/deuterium exchange experiments can give site-specific information on the exposure to or protection from solvent of all residues only if the deuterium label remains fixed during digestion, ionisation and fragmentation of proteins. Using cytochrome c as a model, the authors present evidence that the label is not scrambled during peptic digestion or collision-induced dissociation if the b-ions are monitored, as the mass spectrometry results closely match those observed by NMR methods. 30. Matagne A, Chung EW, Ball LJ, Radford SE, Robinson CV, Dobson CM: The origin of the α-domain intermediate in the folding of hen lysozyme. J Mol Biol 1998, 277:997-1005.
19. Millar AL, Jackson NAC, Dalton H, Jennings KR, Levi M, Wahren B, • Dimmock NJ: Rapid analysis of epitope–paratope interactions between HIV-1 and a 17-amino-acid neutralizing microantibody by electrospray ionization mass spectrometry. Eur J Biochem 1998, 258:164-169. A peptide derived from the gp120 envelope glycoprotein of HIV-1 is seen to interact with a specific antibody. Key residues for the interaction are determined, by digestion of the peptide.
31. Kulkarni SK, Ashcroft AE, Carey M, Masselos D, Robinson CV, Radford SE: A near-native state on the slow refolding pathway of hen lysozyme. Protein Sci 1999, 8:35-44.
20. Chung E, Henriques D, Renzoni D, Zvelebil M, Bradshaw JM, Waksman G, Robinson CV, Ladbury JE: Mass spectrometric and thermodynamic studies reveal the role of water molecules in complexes formed between SH2 domains and tyrosyl phosphopeptides. Structure 1998, 6:1141-1151.
33. van den Berg B, Chung EW, Robinson CV, Dobson CM: • Characterisation of the dominant oxidative folding intermediate of hen lysozyme. J Mol Biol 1999, in press. The refolding of hen egg white lysozyme from a fully unfolded species with reduced cysteine residues is shown to proceed via a stable intermediate with three correctly formed disulfide bonds. The unformed bond is identified by accurate mass measurement by Fourier-transform mass spectrometry (FTMS) followed by digestion of the intermediate and tandem MS of the fragments.
21. Marshall AG, Senko MW, Li WQ, Li M, Dillon S, Guan SH, Logan TM: Protein molecular mass to 1 Da by C-13, N-15 double-depletion and FT-ICR mass spectrometry. J Am Chem Soc 1997, 119:433-434. 22. Shi SDH, Hendrickson CL, Marshall AG: Counting individual sulfur atoms in a protein by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry: experimental resolution of isotropic fine structure in proteins. Proc Natl Acad Sci USA 1998, 95:11532-11537. 23. Chung EW, Nettleton EJ, Morgan CJ, Gross M, Miranker A, Radford SE, Dobson CM, Robinson CV: Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR. Protein Sci 1997, 6:1316-1324. 24. Wang F, Li WQ, Emmett MR, Hendrickson CL, Marshall AG, •• Zhang YL, Wu L, Zhang ZY: Conformational and dynamic changes of Yersinia protein tyrosine phosphatase induced by ligand binding and active site mutation and revealed by H/D exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Biochemistry 1998, 37:15289-15299. Changes in the dynamics of the enzyme are revealed by comparing hydrogen exchange mass spectrometry (MS) data of the free and ligand-bound species. Previous crystal structures comparing ligand-free and ligand-bound proteins reveal a flexible loop that closes around an inhibitor. MS reveals that residues screened by closure of this loop have increased protection from HX, and also that regions of the protein that are very similar in the crystal structures have altered HX protection. 25. Engen JR, Smithgall TE, Gmeiner WH, Smith DL: Comparison of • SH3 and SH2 domain dynamics when expressed alone or in an SH(3+2) construct: the role of protein dynamics in functional regulation. J Mol Biol 1999, 287:645-656. Interdomain interactions are shown to alter the dynamics of the individual domains, reflected in alteration of HX characteristics.
32. Coyle JE, Texter FL, Ashcroft AE, Masselos D, Robinson CV, Radford SE: GroEL accelerates the refolding of hen lysozyme without changing its folding mechanism. Nat Struct Biol 1999, 6:683-690.
34. Tsui V, Garcia C, Cavagnero S, Siuzdak G, Dyson HJ, Wright PE: Quench-flow experiments combined with mass spectrometry show apomyoglobin folds through an obligatory intermediate. Protein Sci 1999, 8:45-49. 35. Forge V, Wijesinha RT, Balbach J, Brew K, Robinson CV, Redfield C, Dobson CM: Rapid collapse and slow structural reorganisation during the refolding of bovine α-lactalbumin. J Mol Biol 1999, 288:673-688. 36. Booth DR, Sunde M, Bellotti V, Robinson CV, Hutchinson WL, Fraser PE, Hawkins PN, Dobson CM, Radford SE, Blake CCF, Pepys MB: Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature 1997, 385:787-793. 37. •
Canet D, Sunde M, Last AM, Miranker A, Spencer A, Robinson CV, Dobson CM: Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behaviour in its disease related variants. Biochemistry 1999, 38:6419-6427. Human lysozyme variants are shown to refold via short-lived intermediates. One of the variants has markedly different refolding kinetics, observed by the increased lifetime of an intermediate state. 38. Nettleton EJ, Sunde M, Lai ZH, Kelly JW, Dobson CM, Robinson CV: • Protein subunit interactions and structural integrity of amyloidogenic transthyretins: evidence from electrospray mass spectrometry. J Mol Biol 1998, 281:553-564. The effect of amyloidogenic mutations on the stability of the transthyretin tetramer is shown by comparing the ease of dissociation of the variants with the wild type.
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39. Vis H, Heinemann U, Dobson CM, Robinson CV: Detection of a •• monomeric intermediate associated with dimerization of protein HU by mass spectrometry. J Am Chem Soc 1998, 120:6427-6428. The stability of the DNA-binding protein HU is shown to increase with salt concentration. Mass spectrometry also reveals an intermediate, partially folded monomer, which is formed at low salt concentration. 40. Vis H, Dobson CM, Robinson CV: Selective association of protein molecules followed by mass spectrometry. Protein Sci 1999, 8:1368-1370. 41. Bothner B, Schneemann A, Marshall D, Reddy V, Johnson JE, •• Siuzdak G: Crystallographically identical virus capsids display different properties in solution. Nat Struct Biol 1999, 6:114-116. The dynamics of crystallographically identical virus structures are markedly different, despite their identical crystal structures. The recombinant virus, containing a random segment of RNA, is both more rapidly digested and more susceptible to covalent modification than the wild type, which contains the correct viral RNA. 42. Lewis JK, Bothner B, Smith TJ, Siuzdak G: Antiviral agent blocks •• breathing of the common cold virus. Proc Natl Acad Sci USA 1998, 95:6774-6778. X-ray analysis shows that the envelope protein VP4 appears to be buried near the core of human rhinovirus 14, yet is amenable to digestion by trypsin.
This shows the large dynamic motions that occur even in this relatively large structure. 43. Rostom AA, Sunde M, Richardson SJ, Schreiber G, Jarvis S, Bateman R, Dobson CM, Robinson CV: Dissection of multi-protein complexes using mass spectrometry — subunit interactions in transthyretin and retinol-binding protein complexes. Proteins 1998, 2(suppl):3-11. 44. Benjamin DR, Robinson CV, Hendrick JP, Hartl FU, Dobson CM: •• Mass spectrometry of ribosomes and ribosomal subunits. Proc Natl Acad Sci USA 1998, 93:7391-7395. Despite its impressive size, the ribosome can be studied by mass spectrometry. Dissociation products reveal which of the subunits interact strongly, and HX shows how flexible these subunits are within the intact complex. 45. Rostom AA, Robinson CV: Detection of the intact GroEL •• chaperonin assembly by mass spectrometry. J Am Chem Soc 1999, 121:4718-4719. The large macromolecular complex of GroEL can be maintained intact in the gas phase. Known connectivities are seen to be preserved upon collisioninduced dissociation of the complex, showing that this method could be used to study species where the links are unknown. 46. Rostom AA, Robinson CV: Disassembly of intact multiprotein complexes in the gas phase. Curr Opin Struct Biol 1999, 9:135-141.
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Protein folding and interactions revealed by mass spectrometry Alexander M Last and Carol V Robinson* Mass spectrometry is capable of examining very large, dynamic proteins and this ability, coupled with its relatively high throughput and low sample requirements, is reflected by its increasing importance for the characterisation of protein structure. Recent developments in mass spectrometry, in particular the refinement of the electrospray process and its coupling with time-of-flight mass analysis, mean that it is poised to contribute not only as a complementary tool but also with a defined role in many areas of chemical biology.
study of noncovalent interactions by MS. The current view is that many more conformations are available to gas-phase ions [6••], depending on the time scale and conditions of the experiment, but that some elements of secondary structure are retained. The overwhelming number of examples, however, where both stoichiometry and even relative affinity of binding diverse ligands to protein targets, provides compelling evidence for preservation of some tertiary structure under carefully controlled conditions.
Addresses Oxford Centre for Molecular Sciences, South Parks Road, Oxford OX1 3QT, UK *e-mail: [email protected]
Initial investigations of protein–ligand complexes using MS indicated an enhanced sensitivity for ionic interactions over hydrophobic ones [7,8]. Numerous examples have been reported of ionic interactions involving metals binding to proteins, including metallothioneins that form intermediates with 4Cu(I) ion clusters before the native 6Cu(I) species [9], and zinc ions bound to hexameric insulin complexes [10]. The refinement of the electrospray process, with the introduction of the nanoflow technique, in which both the size of the needle orifice and the flow rate of the sample are reduced, has led to a smaller droplet size requiring much milder desolvation. This, coupled with the lower voltages in the source, has enabled the survival of weakly associated protein–ligand complexes and allowed the contribution of hydrophobically driven interactions to be addressed [11••]. For example, MS analysis of the binding of aldose reductase to four different inhibitors showed that the strength of binding in the gas phase could be correlated to the electrostatic and hydrogen-bonding interactions defined by X-ray analysis, assuming that the hydrophobic component to the binding energy remained constant [12•].
Current Opinion in Chemical Biology 1999, 3:564–570 1367-5931/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviations FHV Flock House virus FTICR Fourier-transform ion cyclotron resonance HX hydrogen exchange MS mass spectrometry RBP retinol-binding protein TTR transthyretin VLP virus-like particles
Introduction The major incentive behind the design and implementation of new technologies in mass spectrometry (MS) is the drive to obtain increased resolution and mass accuracy, together with an increase in the efficiency of the ionisation processes. In pursuit of these objectives, the introduction of nanoflow electrospray [1] and its marriage with time-offlight [2], and its increased efficiency with Fourier-transform ion cyclotron resonance (FTICR) MS [3], have had a major impact. The introduction of novel combinations such as quadrupole time-of-flight [4] has also extended the tandem capabilities of MS. Our focus in this review is to bring together a diverse range of applications involving electrospray that have arisen largely from these developments. We also highlight the level of structural information available from both individual proteins and their complexes.
Protein–ligand interactions The technique of electrospray produces a stream of highly charged droplets by the continuous flow of a protein solution through a needle maintained at high voltage [5]. A series of evaporation and desorption events produce gas phase ions that are subsequently attracted into the mass spectrometer by electric and pressure gradients. The extent to which the conformation of the gas phase ion produced as a result of this process mirrors that of the protein in solution is an area of major research, not only because of its scientific interest but also because of its impact on the
The ionic interactions between proteins and nucleic acids and the effect of cofactors are readily observed. For example, the trp repressor homodimer binds to a 21-base-pair DNA sequence, yet remains monomeric in the absence of this sequence [13] while the zinc-mediated binding of the nucleocapsid protein (NCp7) to NCp7-psi RNA was not observed in the absence of the metal ion [14]. Metal cofactors have also been investigated in the context of the vitamin D receptor binding to double-stranded DNA [15]. The bacteriophage T4 regA protein in the presence of target sequences of RNA showed stable gas-phase complexes that were resistant to dissociation in the gas phase [16]. Fragmentation of the peptide backbone occurred before the complex was dissociated, providing insight into the RNA-binding site. An alternative MS-based method for identifying DNA-binding sites in a protein has been described and involves chemical derivatisation of the lysine sidechains involved in a protein–DNA interaction [17•]. Small peptide ligands, such as those derived from the bacterial cell wall, and their interactions with the glycopeptide
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Figure 1 Nanoflow electrospray ionisation mass spectra showing the effect of increasing collision energy on the complex formed between the Fyn SH2 domain and the peptide EPQY*EEEPIYL (single letter amino acid code, where Y* is phosphotyrosine). The charge states of each mass spectrum have been converted onto a mass scale and are normalised according to the actual intensities of the spectra. At low cone voltage (35 V) the water-free protein–peptide complex is the dominant species (peak A). Increasing the cone voltage to 80 V disrupts the complex and the apo-protein now predominates (peak B). Further increases in the collision energy (to a cone voltage of 100 V and 140 V) leads to a further disruption of the complex. The water-bound complex, however, is now more intense than the water-free complex (peaks C and D). Dashed lines are used to align the corresponding peaks in each spectrum.
apo-SH2 SH2–EPQY*EEEPIYL 100
Water-bound complex
A
B
C % D 140 100 V) ( ge lta o ev
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antibiotics vancomycin and ristocetin, have been examined by MS and the relative binding affinities assessed [18]. In addition, peptides from HIV-1 were bound to a 17-amino-acid microantibody and the complex was examined using MS; key residues in the HIV peptides were revealed by enzymatic digestion [19 •] — binding decreased dramatically as certain residues were removed, thus revealing the residues required for high-affinity interactions. The binding of peptide mimics of protein ligands to the Src homology 2 (SH2) domain from Src tyrosine kinases was examined by MS. Analysis of the spectra revealed peaks with masses that correspond to the presence of water molecules attached to the apo-protein and peptide-bound complex [20]. The numbers of water molecules bound to the complex in the gas phase correlates well with the tightly bound molecules seen in the crystal structure. In addition, recent evidence has shown that for the SH2 domain from Fyn, three water molecules remain tightly bound even after the energy of collisions is increased. This observation strongly suggests that these water molecules mediate interactions in the ligand-binding interface, conferring an additional stability to the complex even in the gas phase [11••] (Figure 1).
Counting individual atoms An electrospray mass spectrum comprises a series of peaks, known as a ‘charge state series’, from which the mass of a protein can be determined. The width of a charge state arises from the convolution of naturally occurring isotopes within the protein, principally the 13C atoms, and the resolving power of the instrument. By using proteins that have minimal amounts of 13C and 15N, the width of the peak is greatly reduced [21]. Using FTICR, ultra-high resolution was obtained, enabling peaks arising from isotopic fine structure to be separated. For example, using a doubly depleted 13C and 15N sample of the tumour suppressor
13,000 Mass
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protein p16, it was possible to determine the number of sulfur atoms directly from the ultra-high-resolution mass spectrum as 5 ± 0.3 without use of any other information about the protein (Figure 2) [22]. Counting atoms forms the basis of measuring the conformational dynamics of proteins by hydrogen exchange (HX). In this process, a fully protonated protein is diluted Figure 2
∗
∗
# [M+16H]16+
Monoisotopic peak
988.0
988.5
#18O
#34S
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m/z FTICR mass spectrum of p16 tumor suppressor protein showing the monoisotopic peak for the protein and the peaks assigned to the protein containing 34S and 18O atoms. The isotope distribution of the +16 charge state is inset, and an expansion of the labelled peaks is shown. The asterisk represents the peak resulting from the monoisotopic mass and the hash represents the peak resulting from addition of the two Daltons, assigned to a species containing either a single atom of 34S or 18O. From the ratio of the heights of the monoisotopic and the 34S peaks, the number of sulfur atoms in the protein was determined as 5 ± 0.3. Reproduced with permission from Proc Nat Acad Sci USA. m/z, mass/change ratio.
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into a deuterated buffer. All labile protons in sidechains and solvent-exposed backbone amide protons will exchange and the number of deuterons incorporated provides a measure of the conformational dynamics or stability of the protein [23]. When HX is followed by enzymatic digestion and high-performance liquid chromatography (HPLC) separation, it is possible to locate regions of the protein that are protected against exchange with solvent. Using this approach with FTICR analysis, several regions of the variant form of Yersinia protein tyrosine phosphatase were found to be more dynamic than those of its wild type counterpart, and binding of an inhibitor was found to decrease the flexibility of the wild type protein but increase that of the mutant [24••]. This approach has also shown that two types of protein domains, the SH2 and SH3 domains from Hck, have different HX kinetics whether expressed in isolation or as a single polypeptide chain [25•]. Thus the dynamics and solvent accessibility of regions of each domain are altered in the two domain construct, changing the rate at which protons are able to exchange for solvent deuterons. Using a combined NMR and MS approach, cooperative HX in the third domain of the turkey protein ovomucoid showed that the number of amide protons exchanging in a cooperative manner is lower than that predicted by NMR measurements alone [26•].
Site specificity in hydrogen exchange labelling Locating the individual backbone amides that are protected from exchange can be achieved through two distinct methods. The first, mentioned above, employs a combination of enzymatic digestion and HPLC separation of the resulting fragments in which deuterium labelling is preserved. Alternatively, it has been proposed that collision-induced dissociation under high-energy conditions may yield peptide fragments from which HX protection can be deduced. Initial investigations of cytochrome c showed that under the high-energy collision-induced dissociation conditions, deuterons had migrated along the backbone of the protein leading to scrambling of the labelling information [6••]. Interestingly, these observations led to the proposal that under electron capture conditions collision-induced migration of deuterons may be significantly reduced [27,28••]. Moreover, it has recently been demonstrated that under some circumstances it may be possible to use collisioninduced dissociation of peptides to locate deuterons [29••]. HX of native cytochrome c followed by peptic digestion and MS analysis, showed that amino-terminally derived fragments, formed by cleavage across amide linkages, gave rise to retention of deuterium label, while carboxy-terminal fragments lead to hydrogen rearrangements. The apparent conflict between the results from peptides derived from enzymatic digestion and the intact protein presumably arises from the lower enery required to fragment the former.
Observation of folding intermediates The ability of MS to distinguish between populations of molecules with different amounts of deuterium label
enables the study of the heterogeneity of protein folding reactions. Using a pulse labelling method, partially structured species have been identified that incorporate intermediate numbers of deuterons during the folding reaction (i.e. between the number in the fully unfolded and in the native protein). Previous studies of hen egg white lysozyme, a two domain protein with one region having mainly α-helical structure (the α-domain) and the other consisting predominantly of β-sheet (the β-domain), have shown multiple folding pathways when refolding is carried out at pH 5.0. Approximately 30% of molecules are rapidly protected from HX in both domains. The remainder proceeds via a well-defined intermediate corresponding in mass to persistent structure in the α-domains. At high temperature, MS has shown the loss of the α-domain intermediate, whereas high salt concentrations were found to regenerate the properties associated with this intermediate [30]. The effect of metal ions on refolding was found to retard a late step in folding, possibly the docking of two pre-formed domains, whereas refolding at nearer physiological pH shows that all molecules fold via an obligatory intermediate [31]. The interactions of lysozyme folding intermediates with the molecular chaperone GroEL showed that the chaperone interacts with a species late in the folding reaction that was already sufficiently compact to have native-state HX protection [32]. The oxidative refolding of hen lysozyme analysed by enzymatic digestion, FTICR and tandem MS [33•] showed that the last disulfide bond to form was the one linking the two domains, which is consistent with the nonoxidative refolding, where the final docking constitutes a late folding event. Using a combined HX labelling MS approach, the folding pathway of apomyoglobin was also shown to proceed via a discrete intermediate [34]. However, the refolding of the calcium-binding protein bovine α-lactalbumin, which is structurally homologous to the C-type lysozymes, showed no well-defined intermediates by MS. Rather, in its apoform the protein was found to undergo rapid collapse to a molten globule-type intermediate, followed by a slow rearrangement to the native state [35]. The addition of Ca2+ ions to the refolding buffer was found to accelerate this rearrangement to the native state by a factor of two orders of magnitude. HX labelling has also been employed to study the refolding of two variants of human lysozyme that form amyloid fibrils in vivo. Human lysozyme has two naturally occurring variants, Ile56→Thr and Asp67→His, that have been shown, using HX and MS, to have enhanced conformational dynamics relative to their wild type counterpart [36]. In additional experiments, the rates of formation of folding intermediates indicate that the Ile56→Thr variant, which disrupts the interface between the two domains of the protein, decelerates the conversion from a late intermediate to the native state [37 •] (Figure 3). In contrast, the Asp67→His variant has no observable effect on the formation of the native state.
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Protein association Wild type and variant forms of human transthyretin (TTR) can also form amyloid fibrils in vivo. The onset of clinical symptoms (i.e. familial amyloid neuropathy) resulting from amyloid deposition occurs earlier if a variant is present, suggesting that mutation has decreased the stability of the protein. TTR is functional as a homotetramer and by comparing the severity of MS conditions required to disrupt this tetramer it was found that the wild type protein was much more resistant to collision-induced dissociation than either the Leu55→Pro or Val30→Met variants [38•] (Figure 4). Lowering the ionic strength results in a less stable tetramer, whereas addition of thyroxine, the hormone carried by TTR, increases the stability of the intact species.
Figure 3
IIe56-Thr
Wild type
N U
100
N U
I
I
80
60
40
The DNA-binding protein HU is functional as a dimer but remains in a monomeric, unfolded state if present in solution at low salt concentration [39••]. As the ionic strength is increased, the monomer becomes more compact, reflected in the decreased numbers of charges per monomer, and then assembles to the dimer. The HX rates of this protein decrease as ionic strength rises (i.e. as the proportion of the unfolded monomer decreases). In a separate investigation, two structurally similar forms of HU, derived from related bacteria, were able to form a heterodimer when a mixture of the unfolded proteins was maintained at low salt concentration or high temperature. Under these conditions, the unfolded proteins were able to refold and calculation of the binding energies of the homodimers was possible [40].
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2000.0 800.0 500.0 200.0 108.0 38.5 11.3 5.8 3.5 0.0
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Electrospray mass spectra of the refolding of the Ile56→Thr variant and 15N-labelled wild type human lysozymes from 0–2 s. The peaks are labelled U, I and N and represent unfolded, intermediate and native-state populations, respectively. m/z, mass to charge ratio.
Figure 4
(a) Electrospray mass spectra of the wild type TTR tetramer at different cone voltages. At low cone voltages, the predominant species is that of the tetramer (between mass/charge [m/z] ratios of 3,000–4,000), whereas at high cone voltage, only the monomeric species is detected. The mass/charge ratio of the tetramer is markedly different from the monomer due to formation of salt bridges in the intact species reducing the number of ionizable groups, and the
presence of counter-ions in the macromolecular structure. The asterisks denote fragmentation of the peptide backbone at proline 11. (b) The effects of increasing cone voltage on the proportions of tetramer present in the mass spectrum as a percentage of total TTR for wild-type (circle), Val30→Met (square) and Leu55→Pro (diamond). Note that the tetramers formed for both variants are markedly less stable than that formed with the wild-type protein.
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Subunit packing in macromolecular complexes One of the most exciting applications of MS has been to show that crystallographically identical virus structures do not necessarily share identical dynamics [41••]. Flock House virus (FHV) reproduction is altered depending on the host organism. In Drosophila, the correct viral RNA is packaged, forming an FHV particle; in baculovirus, however, virus-like particles (VLPs), containing cellular RNA, are formed. X-ray crystallographic analyses of FHV and VLPs show that the capsid structures are identical. When the FHV and VLPs are exposed to enzymatic digestion and covalent modification, however, the rates of reaction were found to be faster for the VLPs than for the FHV, suggesting that the viral RNA contributes to the overall stability of the capsid structure. Similarly, the VP4 coat protein of human rhinovirus 14 is located beneath the surface of the capsid, yet is readily digested by trypsin [42••]. This result is interpreted in terms of ‘breathing’ of the capsid, thus allowing trypsin to permeate and interact with a protein not thought to be exposed to solvent. Addition of an antiviral drug reduces the amount of digestion products formed and hence reduces the fluctuations of the capsid surface. As well as being the thyroxine carrier in blood, TTR also forms a physiologically important complex with retinolbinding protein (RBP). Several TTR–RBP complexes, with differing stoichiometries, are feasible. Simulation of the overlapping charge states allows determination of the relative amounts of the different complexes with one or two RBP molecules bound. From this simulation, dissociation constants were calculated and the results show that binding of the first RBP has negative cooperativity for the binding of the second [43]. Moreover, dissociation in the gas phase was used to identify the connectivities between the different subunits. This approach has also been employed in the study of ribosomes from Escherichia coli. Despite the size and complexity of the 2.3 MDa particle, well-resolved spectra of the dissociation products have been obtained, enabling the topology of the particle to be examined [44••]. A dramatic example of both the maintenance and disassembly of macromolecular complexes is that of an 800 kDa complex of the molecular chaperone GroEL from E. coli [45••,46]. Well-defined charge states for the intact complex were observed at a mass/charge value of 10,000, from which the mass was calculated as 803,742 ± 616 Da. Collisioninduced dissociation of this macromolecular complex showed that the symmetry of the subunit packing in the two heptameric rings was retained even in the gas phase.
Conclusions The phenomenal growth of mass spectrometry in recent years is due not only to its capabilities in defining the primary structure of proteins from electrophoretic gels, but also because of its applications to noncovalent interactions and folding, as described herein. The numerous examples where both stoichiometry and relative affinity in the bind-
ing of protein ligand and macromolecular complexes correlates with solution conditions puts the methodology beyond reasonable doubt. As MS enters a new era — studying the assembly of macromolecular complexes — the many and varied investigations of protein conformation and interactions lay the foundation for the use of MS in the wider context of structural biology.
Acknowledgements We acknowledge, with thanks, many helpful discussions with the Robinson MS group and with Chris Dobson. This contribution was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), Engineering and Physical Science Research Council (EPSRC), and the Medical Research Council. Carol Robinson also acknowledges support from the Royal Society.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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McLafferty FW, Guan ZQ, Haupts U, Wood TD, Kelleher NL: Gaseous conformational structures of cytochrome c. J Am Chem Soc 1998, 120:4732-4740. The location of deuterium label in cytochrome c was examined by collisioninduced dissociation and, despite extensive scrambling of labile deuterons, the results show that four small helical regions are maintained, even in the most open conformations. 7.
Robinson CV, Chung EW, Kragelund BB, Knudsen J, Aplin RT, Poulsen FM, Dobson CM: Probing the nature of non-covalent interactions by mass spectrometry. A study of protein-CoA ligand binding and assembly. J Am Chem Soc 1996, 118:8646-8653.
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Jensen LT, Peltier JM, Winge DR: Identification of a four copper folding intermediate in mammalian copper metallothionein by electrospray ionization mass spectrometry. J Biol Inorg Chem 1998, 3:627-631.
10. Fabris D, Fenselau C: Characterisation of allosteric insulin hexamers by electrospray ionization mass spectrometry. Anal Chem 1999, 71:384-387. 11. Chung EW, Henriques D, Renzoni D, Ladbury JE, Robinson CV: •• Probing the nature of interactions in SH2 binding interfaces — evidence from electrospray mass spectrometry. Protein Sci 1999, in press. This paper shows that under high-energy collision conditions up to three water molecules remain bound to the complex, presumably as a result of hydrogen bonding interactions in the ligand binding interface. 12. Rogniaux H, Van Dorsselaer A, Barth P, Biellmann JF, Barbanton J, • van Zandt M, Chevrier B, Howard E, Mitschler A, Potier N et al.: Binding of aldose reductase inhibitors: correlation of
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crystallographic and mass spectrometric studies. J Am Soc Mass Spectrom 1999, 10:635-647. In this study, both X-ray crystallography and mass spectrometry were applied to aldose reductase and four potential inhibitors and the results were used to demonstrate the complementarity between the two techniques. 13. Potier N, Donald LJ, Chernushevich I, Ayed A, Ens W, Arrowsmith CH, Standing KG, Duckworth HW: Study of a noncovalent trp repressor:DNA operator complex by electrospray time-of-flight mass spectrometry. Protein Sci 1998, 7:1388-1395. 14. Loo JA, Holler TP, Foltin SK, McConnell P, Banotai CA, Horne NM, Mueller WT, Stevenson TI, Mack DP: Application of electrospray ionization mass spectrometry for studying human immunodefeciency virus protein complexes. Proteins 1998, 2(suppl):28-37. 15. Veenstra TD, Benson LM, Craig TA, Tomlinson AJ, Kumar R, Naylor S: Metal mediated sterol receptor–DNA complex association and dissociation by electrospray ionization mass spectrometry. Nat Biotechnol 1998, 16:262-266. 16. Liu CL, Tolic LP, Hofstadler SA, Harms AC, Smith RD, Kang CH, Sinha N: Probing regA/RNA interactions using electrospray ionization Fourier transform ion cyclotron resonance-mass spectrometry. Anal Biochem 1998, 262:67-76. 17. •
Chen JW, Smith DL, Griep MA: The role of the 6 lysines and the terminal amine of Escherichia coli single-strand binding protein in its binding of single-stranded DNA. Protein Sci 1998, 7:1781-1788. The authors present a method for covalent modification of lysine residues allowing differential labelling to probe the accessibility of the sidechain amino group and their interactions with DNA. 18. Jorgensen TJD, Roepstorff P, Heck AJR: Direct determination of solution binding constants for noncovalent complexes between bacterial cell wall peptide analogues and vancomycin group antibiotics by electrospray ionization mass spectrometry. Anal Chem 1998, 70:4427-4432.
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26. Arrington CB, Teesch LM, Robertson AD: Defining protein • ensembles with native state NH exchange: kinetics of interconversion and cooperative units from combined NMR and MS analysis. J Mol Biol 1999, 285:1265-1275. NMR-derived HX data of the turkey ovomucoid domain are used to simulate peak widths during mass spectrometry (MS) exchange. Observed MS peaks do not match these, suggesting that the extent of cooperativity is more limited than suggested using NMR. 27.
Zubarev RA, Kelleher Nl, McLafferty FW: Electron capture dissociation of multiply charged protein cations. A nonergodic process. J Am Chem Soc 1998, 120:3265-3266.
28. Zubarev RA, Kruger NA, Frediksson EK, Lewis MA, Horn DM, •• Carpenter BK, McLafferty FW: Electron capture dissociation of gaseous multiply charged proteins is favoured at disulphide bonds and other sites of high hydrogen atom affinity. J Am Chem Soc 1999, 121:2857-2862. The electron capture dissociation method was shown to generate preferential cleavage of the backbone at disulfide bonds, in contrast to excitation of the ions by photons or low-energy collisions. 29. Deng YZ, Pan H, Smith DL: Selective isotope labeling •• demonstrates that hydrogen exchange at individual peptide amide linkages can be determined by collision-induced dissociation mass spectrometry. J Am Chem Soc 1999, 121:1966-1967. Hydrogen/deuterium exchange experiments can give site-specific information on the exposure to or protection from solvent of all residues only if the deuterium label remains fixed during digestion, ionisation and fragmentation of proteins. Using cytochrome c as a model, the authors present evidence that the label is not scrambled during peptic digestion or collision-induced dissociation if the b-ions are monitored, as the mass spectrometry results closely match those observed by NMR methods. 30. Matagne A, Chung EW, Ball LJ, Radford SE, Robinson CV, Dobson CM: The origin of the α-domain intermediate in the folding of hen lysozyme. J Mol Biol 1998, 277:997-1005.
19. Millar AL, Jackson NAC, Dalton H, Jennings KR, Levi M, Wahren B, • Dimmock NJ: Rapid analysis of epitope–paratope interactions between HIV-1 and a 17-amino-acid neutralizing microantibody by electrospray ionization mass spectrometry. Eur J Biochem 1998, 258:164-169. A peptide derived from the gp120 envelope glycoprotein of HIV-1 is seen to interact with a specific antibody. Key residues for the interaction are determined, by digestion of the peptide.
31. Kulkarni SK, Ashcroft AE, Carey M, Masselos D, Robinson CV, Radford SE: A near-native state on the slow refolding pathway of hen lysozyme. Protein Sci 1999, 8:35-44.
20. Chung E, Henriques D, Renzoni D, Zvelebil M, Bradshaw JM, Waksman G, Robinson CV, Ladbury JE: Mass spectrometric and thermodynamic studies reveal the role of water molecules in complexes formed between SH2 domains and tyrosyl phosphopeptides. Structure 1998, 6:1141-1151.
33. van den Berg B, Chung EW, Robinson CV, Dobson CM: • Characterisation of the dominant oxidative folding intermediate of hen lysozyme. J Mol Biol 1999, in press. The refolding of hen egg white lysozyme from a fully unfolded species with reduced cysteine residues is shown to proceed via a stable intermediate with three correctly formed disulfide bonds. The unformed bond is identified by accurate mass measurement by Fourier-transform mass spectrometry (FTMS) followed by digestion of the intermediate and tandem MS of the fragments.
21. Marshall AG, Senko MW, Li WQ, Li M, Dillon S, Guan SH, Logan TM: Protein molecular mass to 1 Da by C-13, N-15 double-depletion and FT-ICR mass spectrometry. J Am Chem Soc 1997, 119:433-434. 22. Shi SDH, Hendrickson CL, Marshall AG: Counting individual sulfur atoms in a protein by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry: experimental resolution of isotropic fine structure in proteins. Proc Natl Acad Sci USA 1998, 95:11532-11537. 23. Chung EW, Nettleton EJ, Morgan CJ, Gross M, Miranker A, Radford SE, Dobson CM, Robinson CV: Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR. Protein Sci 1997, 6:1316-1324. 24. Wang F, Li WQ, Emmett MR, Hendrickson CL, Marshall AG, •• Zhang YL, Wu L, Zhang ZY: Conformational and dynamic changes of Yersinia protein tyrosine phosphatase induced by ligand binding and active site mutation and revealed by H/D exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Biochemistry 1998, 37:15289-15299. Changes in the dynamics of the enzyme are revealed by comparing hydrogen exchange mass spectrometry (MS) data of the free and ligand-bound species. Previous crystal structures comparing ligand-free and ligand-bound proteins reveal a flexible loop that closes around an inhibitor. MS reveals that residues screened by closure of this loop have increased protection from HX, and also that regions of the protein that are very similar in the crystal structures have altered HX protection. 25. Engen JR, Smithgall TE, Gmeiner WH, Smith DL: Comparison of • SH3 and SH2 domain dynamics when expressed alone or in an SH(3+2) construct: the role of protein dynamics in functional regulation. J Mol Biol 1999, 287:645-656. Interdomain interactions are shown to alter the dynamics of the individual domains, reflected in alteration of HX characteristics.
32. Coyle JE, Texter FL, Ashcroft AE, Masselos D, Robinson CV, Radford SE: GroEL accelerates the refolding of hen lysozyme without changing its folding mechanism. Nat Struct Biol 1999, 6:683-690.
34. Tsui V, Garcia C, Cavagnero S, Siuzdak G, Dyson HJ, Wright PE: Quench-flow experiments combined with mass spectrometry show apomyoglobin folds through an obligatory intermediate. Protein Sci 1999, 8:45-49. 35. Forge V, Wijesinha RT, Balbach J, Brew K, Robinson CV, Redfield C, Dobson CM: Rapid collapse and slow structural reorganisation during the refolding of bovine α-lactalbumin. J Mol Biol 1999, 288:673-688. 36. Booth DR, Sunde M, Bellotti V, Robinson CV, Hutchinson WL, Fraser PE, Hawkins PN, Dobson CM, Radford SE, Blake CCF, Pepys MB: Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature 1997, 385:787-793. 37. •
Canet D, Sunde M, Last AM, Miranker A, Spencer A, Robinson CV, Dobson CM: Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behaviour in its disease related variants. Biochemistry 1999, 38:6419-6427. Human lysozyme variants are shown to refold via short-lived intermediates. One of the variants has markedly different refolding kinetics, observed by the increased lifetime of an intermediate state. 38. Nettleton EJ, Sunde M, Lai ZH, Kelly JW, Dobson CM, Robinson CV: • Protein subunit interactions and structural integrity of amyloidogenic transthyretins: evidence from electrospray mass spectrometry. J Mol Biol 1998, 281:553-564. The effect of amyloidogenic mutations on the stability of the transthyretin tetramer is shown by comparing the ease of dissociation of the variants with the wild type.
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39. Vis H, Heinemann U, Dobson CM, Robinson CV: Detection of a •• monomeric intermediate associated with dimerization of protein HU by mass spectrometry. J Am Chem Soc 1998, 120:6427-6428. The stability of the DNA-binding protein HU is shown to increase with salt concentration. Mass spectrometry also reveals an intermediate, partially folded monomer, which is formed at low salt concentration. 40. Vis H, Dobson CM, Robinson CV: Selective association of protein molecules followed by mass spectrometry. Protein Sci 1999, 8:1368-1370. 41. Bothner B, Schneemann A, Marshall D, Reddy V, Johnson JE, •• Siuzdak G: Crystallographically identical virus capsids display different properties in solution. Nat Struct Biol 1999, 6:114-116. The dynamics of crystallographically identical virus structures are markedly different, despite their identical crystal structures. The recombinant virus, containing a random segment of RNA, is both more rapidly digested and more susceptible to covalent modification than the wild type, which contains the correct viral RNA. 42. Lewis JK, Bothner B, Smith TJ, Siuzdak G: Antiviral agent blocks •• breathing of the common cold virus. Proc Natl Acad Sci USA 1998, 95:6774-6778. X-ray analysis shows that the envelope protein VP4 appears to be buried near the core of human rhinovirus 14, yet is amenable to digestion by trypsin.
This shows the large dynamic motions that occur even in this relatively large structure. 43. Rostom AA, Sunde M, Richardson SJ, Schreiber G, Jarvis S, Bateman R, Dobson CM, Robinson CV: Dissection of multi-protein complexes using mass spectrometry — subunit interactions in transthyretin and retinol-binding protein complexes. Proteins 1998, 2(suppl):3-11. 44. Benjamin DR, Robinson CV, Hendrick JP, Hartl FU, Dobson CM: •• Mass spectrometry of ribosomes and ribosomal subunits. Proc Natl Acad Sci USA 1998, 93:7391-7395. Despite its impressive size, the ribosome can be studied by mass spectrometry. Dissociation products reveal which of the subunits interact strongly, and HX shows how flexible these subunits are within the intact complex. 45. Rostom AA, Robinson CV: Detection of the intact GroEL •• chaperonin assembly by mass spectrometry. J Am Chem Soc 1999, 121:4718-4719. The large macromolecular complex of GroEL can be maintained intact in the gas phase. Known connectivities are seen to be preserved upon collisioninduced dissociation of the complex, showing that this method could be used to study species where the links are unknown. 46. Rostom AA, Robinson CV: Disassembly of intact multiprotein complexes in the gas phase. Curr Opin Struct Biol 1999, 9:135-141.