Collisional and Coulombic Unfolding of Gas‐Phase Proteins: High Correlation to Their Domain Structures in Solution

The three‐dimensional structures adopted by proteins are predicated by their many biological functions. Mass spectrometry has played a rapidly expanding role in protein structure discovery, enabling the generation of models for both proteins and their higher‐order assemblies. While important coursed‐grained insights have been generated, relatively few examples exist where mass spectrometry has been successfully applied to the characterization of protein tertiary structure. Here, we demonstrate that gas‐phase unfolding can be used to determine the number of autonomously folded domains within monomeric proteins. Our ion mobility‐mass spectrometry data highlight a strong, positive correlation between the number of protein unfolding transitions observed in the gas phase and the number of known domains within a group of sixteen proteins ranging from 8–78 kDa. This correlation and its potential uses for structural biology is discussed.     

Proteins, lipids, and water in the gas phase

Evidence from mass‐spectrometry experiments and molecular dynamics simulations suggests that it is possible to transfer proteins, or in general biomolecular aggregates, from solution to the gas‐phase without grave impact on the structure. If correct, this allows interpretation of such experiments as a probe of physiological behavior. Here, we survey recent experimental results from mass spectrometry and ion‐mobility spectroscopy and combine this with observations based on molecular dynamics simulation, in order to give a comprehensive overview of the state of the art in gas‐phase studies. We introduce a new concept in protein structure analysis by determining the fraction of the theoretical possible numbers of hydrogen bonds that are formed in solution and in the gas‐phase. In solution on average 43% of the hydrogen bonds is realized, while in vacuo this fraction increases to 56%. The hydrogen bonds stabilizing the secondary structure (α‐helices, β‐sheets) are maintained to a large degree, with additional hydrogen bonds occurring when side chains make new hydrogen bonds to rest of the protein rather than to solvent. This indicates that proteins that are transported to the gas phase in a native‐like manner in many cases will be kinetically trapped in near‐physiological structures. Simulation results for lipid‐ and detergent‐aggregates and lipid‐coated (membrane) proteins in the gas phase are discussed, which in general point to the conclusion that encapsulating proteins in “something” aids in the conservation of native‐like structure. Isolated solvated micelles of cetyl‐tetraammonium bromide quickly turn into reverse micelles whereas dodecyl phosphocholine micelles undergo much slower conversions, and do not quite reach a reverse micelle conformation within 100 ns.

     

Native mass spectrometry—A valuable tool in structural biology

Proteins and the complexes they form with their ligands are the players of cellular action. Their function is directly linked with their structure making the structural analysis of protein‐ligand complexes essential. Classical techniques of structural biology include X‐ray crystallography, nuclear magnetic resonance spectroscopy and recently distinguished cryo‐electron microscopy. However, protein‐ligand complexes are often dynamic and heterogeneous and consequently challenging for these techniques. Alternative approaches are therefore needed and gained importance during the last decades. One alternative is native mass spectrometry, which is the analysis of intact protein complexes in the gas phase. To achieve this, sample preparation and instrument conditions have to be optimised. Native mass spectrometry then reveals stoichiometry, protein interactions and topology of protein assemblies. Advanced techniques such as ion mobility and high‐resolution mass spectrometry further add to the range of applications and deliver information on shape and microheterogeneity of the complexes. In this tutorial, we explain the basics of native mass spectrometry including sample requirements, instrument modifications and interpretation of native mass spectra. We further discuss the developments of native mass spectrometry and provide example spectra and applications.     

Local Transient Unfolding of Native State PAI‐1 Associated with Serpin Metastability

The metastability of the native fold makes serpin (serine protease inhibitor) proteins prone to pathological conformational change, often by insertion of an extra β‐strand into the central β‐sheet A. How this insertion is made possible is a hitherto unresolved question. By the use of advanced hydrogen/deuterium‐exchange mass spectrometry (HDX‐MS) it is shown that the serpin plasminogen activator inhibitor 1 (PAI‐1) transiently unfolds under native condition, on a second‐to‐minute time scale. The unfolding regions comprise β‐strand 5A as well as the underlying hydrophobic core, including β‐strand 6B and parts of helices A, B, and C. Based thereon, a mechanism is proposed by which PAI‐1 makes transitions through progressively more unfolded states along the reaction coordinate to the inactive, so‐called latent form. Our results highlight the profound utility of HDX‐MS in detecting sparsely populated, transiently unfolded protein states.     

Analyzing slowly exchanging protein conformations by ion mobility mass spectrometry: study of the dynamic equilibrium of prolyl oligopeptidase

Ion mobility mass spectrometry (IMMS) is a biophysical technique that allows the separation of isobaric species on the basis of their size and shape. The high separation capacity, sensitivity and relatively fast time scale measurements confer IMMS great potential for the study of proteins in slow (µs–ms) conformational equilibrium in solution. However, the use of this technique for examining dynamic proteins is still not generalized. One of the major limitations is the instability of protein ions in the gas phase, which raises the question as to what extent the structures detected reflect those in solution. Here, we addressed this issue by analyzing the conformational landscape of prolyl oligopeptidase (POP) – a model of a large dynamic enzyme in the µs–ms range – by native IMMS and compared the results obtained in the gas phase with those obtained in solution. In order to interpret the experimental results, we used theoretical simulations. In addition, the stability of POP gaseous ions was explored by charge reduction and collision‐induced unfolding experiments. Our experiments disclosed two species of POP in the gas phase, which correlated well with the open and closed conformations in equilibrium in solution; moreover, a gas‐phase collapsed form of POP was also detected. Therefore, our findings not only support the potential of IMMS for the study of multiple co‐existing conformations of large proteins in slow dynamic equilibrium in solution but also stress the need for careful data analysis to avoid artifacts. Copyright © 2016 John Wiley & Sons, Ltd.     

Monitoring Backbone Hydrogen‐Bond Formation in β‐Barrel Membrane Protein Folding

β‐barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three‐dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β‐strands. Here, we employ hydrogen–deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue‐specific kinetics of interstrand hydrogen‐bond formation were found to be uniform in the entire β‐barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long‐lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate‐limiting transition state and thus appears cooperative on the overall folding time scale.     

Determination of three kinds of banned drugs in milk powder by hybrid solid‐phase extraction purification combined with gas chromatography and mass spectrometry

A gradient clean‐up method for the quantification of five kinds of banned drugs (two hormones, two sedatives, and one chloramphenicol) in milk powder was developed. We used the combination of solid‐phase extraction purification with gas chromatography and mass spectrometry. Milk powder was initially hydrolyzed by β‐glucuronidase/arylsulfatase, and then the hydrolyzed solution was concentrated and purified using a C₈ and cation resin solid‐phase extraction column. To isolate hormones and chloramphenicol drugs, products from the previous step were diluted with methanol and further purified using a silica and diatomite solid‐phase extraction column. After derivatization, the drugs were analyzed by gas chromatography with mass spectrometry, and the hydrolyzed solution was diluted with 5% ammoniated methanol to purify sedatives before gas chromatography with mass spectrometry analysis. Results showed that after adding the banned drugs at concentrations of 0.3–10.0 μg/kg, the average recovery range was 78.2–97.3% with relative standard deviations of 5.3–12.5%. The limit of quantification of the banned drugs (S/N ≥ 10) was 0.3–5.0 μg/kg, whereas the limit of detection (S/N ≥ 3) was 0.1–2.0 μg/kg. The solid‐phase extraction gradient purification system was simple, rapid, and accurate, and could satisfy the detection requirements of hormone, sedatives, and chloramphenicol drugs when used together with gas chromatography and mass spectrometry.     

Gas‐phase behavior of noncovalent transmembrane segment complexes

Specific helix oligomerization between transmembrane segments (TMSs) is often promoted by motifs like GxxxG. Disruption of this motif in the transmembrane segments of vesicular stomatitis virus G‐protein and of glycophorin A results in a reduced dimerization level studied by in vivo systems like ToxR. This paper reports the influence of sequence motifs like GxxxG in solution and the gas phase. The transmembrane segments may behave differently in the gas and liquid phase, because of the absence of surrounding solvent molecules in the gas phase. Comparison of experiments depending on peptide properties performed in the gas and liquid phase discloses that the peptides retain ‘some memory’ of their liquid‐phase structure in the gas phase. A direct correlation has been found between helicity in solution as determined by circular dichroism and dimerization in the gas phase monitored by electrospray mass spectrometry. These results show that a proper folding in solution is required for oligomerization. On the other hand, sequence‐specific oligomerization depending on the GxxxG motif was not observed with the mass spectrometric detection. Further on, neither concentration‐dependent complex studies nor studies regarding complex stability in the gas phase – via collision‐induced dissociation (CID) – led to sequence‐specific differences. Finally, the findings show that in mass spectrometric measurements noncovalent interactions of studied TMSs is rather more dependent on the secondary structure and proper folding than on their primary structure. Copyright © 2008 John Wiley & Sons, Ltd.     

Probing the Lipid Annular Belt by Gas‐Phase Dissociation of Membrane Proteins in Nanodiscs

Interactions between membrane proteins and lipids are often crucial for structure and function yet difficult to define because of their dynamic and heterogeneous nature. Here, we use mass spectrometry to demonstrate that membrane protein oligomers ejected from nanodiscs in the gas phase retain large numbers of lipid interactions. The complex mass spectra that result from gas‐phase dissociation were assigned using a Bayesian deconvolution algorithm together with mass defect analysis, allowing us to count individual lipid molecules bound to membrane proteins. Comparison of the lipid distributions measured by mass spectrometry with molecular dynamics simulations reveals that the distributions correspond to distinct lipid shells that vary according to the type of protein–lipid interactions. Our results demonstrate that nanodiscs offer the potential for native mass spectrometry to probe interactions between membrane proteins and the wider lipid environment.     

Travelling wave ion mobility mass spectrometry studies of protein structure: biological significance and comparison with X-ray crystallography and nuclear magnetic resonance spectroscopy measurements

The three-dimensional conformation of a protein is central to its biological function. The characterisation of aspects of three-dimensional protein structure by mass spectrometry is an area of much interest as the gas-phase conformation, in many instances, can be related to that of the solution phase. Travelling wave ion mobility mass spectrometry (TWIMS) was used to investigate the biological significance of gas-phase protein structure. Protein standards were analysed by TWIMS under denaturing and near-physiological solvent conditions and cross-sections estimated for the charge states observed. Estimates of collision cross-sections were obtained with reference to known standards with published cross-sections. Estimated cross-sections were compared with values from published X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy structures. The cross-section measured by ion mobility mass spectrometry varies with charge state, allowing the unfolding transition of proteins in the gas phase to be studied. Cross-sections estimated experimentally for proteins studied, for charge states most indicative of native structure, are in good agreement with measurements calculated from published X-ray and NMR structures. The relative stability of gas-phase structures has been investigated, for the proteins studied, based on their change in cross-section with increase in charge. These results illustrate that the TWIMS approach can provide data on three-dimensional protein structures of biological relevance.     

Cross‐Linking/Mass Spectrometry for Studying Protein Structures and Protein–Protein Interactions: Where Are We Now and Where Should We Go from Here?

Structural mass spectrometry (MS) is gaining increasing importance for deriving valuable three‐dimensional structural information on proteins and protein complexes, and it complements existing techniques, such as NMR spectroscopy and X‐ray crystallography. Structural MS unites different MS‐based techniques, such as hydrogen/deuterium exchange, native MS, ion‐mobility MS, protein footprinting, and chemical cross‐linking/MS, and it allows fundamental questions in structural biology to be addressed. In this Minireview, I will focus on the cross‐linking/MS strategy. This method not only delivers tertiary structural information on proteins, but is also increasingly being used to decipher protein interaction networks, both in vitro and in vivo. Cross‐linking/MS is currently one of the most promising MS‐based approaches to derive structural information on very large and transient protein assemblies and intrinsically disordered proteins.     

The Role of the Detergent Micelle in Preserving the Structure of Membrane Proteins in the Gas Phase

Despite the growing importance of the mass spectrometry of membrane proteins, it is not known how their transfer from solution into vacuum affects their stability and structure. To address this we have carried out a systematic investigation of ten membrane proteins solubilized in different detergents and used mass spectrometry to gain physicochemical insight into the mechanism of their ionization and desolvation. We show that the chemical properties of the detergents mediate the charge state, both during ionization and detergent removal. Using ion mobility mass spectrometry, we monitor the conformations of membrane proteins and show how the surface charge density dictates the stability of folded states. We conclude that the gas‐phase stability of membrane proteins is increased when a greater proportion of their surface is lipophilic and is consequently protected by the physical presence of the micelle.     

Egg White Varnishes on Ancient Paintings: A Molecular Connection to Amyloid Proteins

For about 400 years, egg white was used to coat and protect paintings without detailed understanding of its molecular properties. A molecular basis is provided for its advantageous properties and one of its protective properties is demonstrated with oxygen transport behavior. Compared to the native secondary structure of ovalbumin in solution of circa 33 % α‐helix and β‐sheet, attenuated total reflection–FTIR (ATR‐FTIR) spectra showed a 73 % decrease of α‐helix content and a 44 % increase of β‐sheet content over eight days. The data suggest that the final coating of dissolved ovalbumin from egg white after long exposure to air, which is hydrophobic, comprises mostly β‐sheet content (ca. 50 %), which is predicted to be the lowest‐energy structure of proteins and close to that found in amyloid fibrils. Coating a synthetic polytetrafluoroethylene membrane with multiple layers of egg white decreased oxygen diffusion by 50 % per layer with a total decrease of almost 100 % for four layers.     

Homogeneous Human Complex‐Type Oligosaccharides in Correctly Folded Intact Glycoproteins: Evaluation of Oligosaccharide Influence On Protein Folding,Stability, and Conformational Properties

The N‐glycosylation of proteins is generated at the consensus sequence NXS/T (where X is any amino acid except proline) by the biosynthetic process, and occurs in the endoplasmic reticulum and Golgi apparatus. In order to investigate the influence of human complex‐type oligosaccharides on counterpart protein conformation, crambin and ovomucoide, which consist of 46 and 56 amino acid residues, respectively, were selected for synthesis of model glycoproteins. These small glycoproteins were intentionally designed to be glycosylated at the α‐helix (crambin: 8 position), β‐sheet (crambin: 2 position) and loop position between the antiparallel β‐sheets (ovomucoide: 28 position), and were synthesized by using a peptide‐segment coupling strategy. After preparation of these glycosylated polypeptide chains, protein folding experiments were performed under redox conditions by using cysteine–cystine. Although the small glycoproteins bearing intentional glycosylation at the α‐helix and β‐sheet exhibited a suitable folding process, glycosylation at the loop position between the antiparallel β‐strands caused multiple products. The conformational differences in the isolated homogeneous glycoproteins compared with non‐glycosylated counterparts were evaluated by circular dichroism (CD) and NMR spectroscopy. These analyses suggested that this intentional N‐glycosylation did not result in large conformational changes in the purified protein structures, including the case of glycosylation at the loop position between the antiparallel β‐strands. In addition to these experiments, the conformational properties of three glycoproteins were evaluated by CD spectroscopy under different temperatures. The oligosaccharides on the protein surface fluctuated considerably; this was dependent on the increase in the solution temperature and was thought to disrupt the protein tertiary structure. Based on the measurement of the CD spectra, however, the glycoproteins bearing three disulfide bonds did not exhibit any change in their protein tertiary structure. These results suggest that the oligosaccharide conformational fluctuations were not disruptive to protein tertiary structure, and the tertiary structure of glycoproteins might be stabilized by the disulfide bond network.     

Quantum mechanical studies on model α‐pleated sheets

Pauling and Corey proposed a pleated‐sheet configuration, now called α‐sheet, as one of the protein secondary structures in addition to α‐helix and β‐sheet. Recently, it has been suggested that α‐sheet is a common feature of amyloidogenic intermediates. We have investigated the stability of antiparallel β‐sheet and two conformations of α‐sheet in solution phase using the density functional theoretical method. The peptides are modeled as two‐strand acetyl‐(Ala)₂‐N‐methylamine. Using stages of geometry optimization and single point energy calculation at B3LYP/cc‐pVTZ//B3LYP/6‐31G* level and including zero‐point energies, thermal, and entropic contribution, we have found that β‐sheet is the most stable conformation, while the α‐sheet proposed by Pauling and Corey has 13.6 kcal/mol higher free energy than the β‐sheet. The α‐sheet that resembles the structure observed in molecular dynamics simulations of amyloidogenic proteins at low pH becomes distorted after stages of geometry optimization in solution. Whether the α‐sheets with longer chains would be increasingly favorable in water relative to the increase in internal energy of the chain needs further investigation. Different from the quantum mechanics results, AMBER parm94 force field gives small difference in solution phase energy between α‐sheet and β‐sheet. The predicted amide I IR spectra of α‐sheet shows the main band at higher frequency than β‐sheet. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

Micelle‐Triggered β‐Hairpin to α‐Helix Transition in a 14‐Residue Peptide from a Choline‐Binding Repeat of the Pneumococcal Autolysin LytA

Choline‐binding modules (CBMs) have a ββ‐solenoid structure composed of choline‐binding repeats (CBR), which consist of a β‐hairpin followed by a short linker. To find minimal peptides that are able to maintain the CBR native structure and to evaluate their remaining choline‐binding ability, we have analysed the third β‐hairpin of the CBM from the pneumococcal LytA autolysin. Circular dichroism and NMR data reveal that this peptide forms a highly stable native‐like β‐hairpin both in aqueous solution and in the presence of trifluoroethanol, but, strikingly, the peptide structure is a stable amphipathic α‐helix in both zwitterionic (dodecylphosphocholine) and anionic (sodium dodecylsulfate) detergent micelles, as well as in small unilamellar vesicles. This β‐hairpin to α‐helix conversion is reversible. Given that the β‐hairpin and α‐helix differ greatly in the distribution of hydrophobic and hydrophilic side chains, we propose that the amphipathicity is a requirement for a peptide structure to interact and to be stable in micelles or lipid vesicles. To our knowledge, this “chameleonic” behaviour is the only described case of a micelle‐induced structural transition between two ordered peptide structures.     

Folding of the Tau Protein on Microtubules

Microtubules are regulated by microtubule‐associated proteins. However, little is known about the structure of microtubule‐associated proteins in complex with microtubules. Herein we show that the microtubule‐associated protein Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules. While Tau is highly flexible in solution and adopts a β‐sheet structure in amyloid fibrils, in complex with microtubules the conserved hexapeptides at the beginning of the Tau repeats two and three convert into a hairpin conformation. Thus, binding to microtubules stabilizes a unique conformation in Tau.     

Probing the hydrophobic effect of noncovalent complexes by mass spectrometry

The study of noncovalent interactions by mass spectrometry has become an active field of research in recent years. The role of the different noncovalent intermolecular forces is not yet fully understood since they tend to be modulated upon transfer into the gas phase. The hydrophobic effect, which plays a major role in protein folding, adhesion of lipid bilayers, etc., is absent in the gas phase. Here, noncovalent complexes with different types of interaction forces were investigated by mass spectrometry and compared with the complex present in solution. Creatine kinase (CK), glutathione S-transferase (GST), ribonuclease S (RNase S), and leucine zipper (LZ), which have dissociation constants in the nM range, were studied by native nanoelectrospray mass spectrometry (nanoESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking (XL). Complexes interacting with hydrogen bonds survived the transfer into gas phase intact and were observed by nanoESI-MS. Complexes that are bound largely by the hydrophobic effect in solution were not detected or only at very low intensity. Complexes with mixed polar and hydrophobic interactions were detected by nanoESI-MS, most likely due to the contribution from polar interactions. All noncovalent complexes could easily be studied by XL MALDI-MS, which demonstrates that the noncovalently bound complexes are conserved, and a real “snap-shot” of the situation in solution can be obtained.     

Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation

Electrospray ionization mass spectrometry (ESI-MS) can be used to monitor conformational changes of proteins in solution based on the charge state distribution (CSD) of the corresponding gas-phase ions, although relatively few studies of acidic proteins have been reported. Here, we have compared the CSD and solution structure of recombinant Vibrio harveyi acyl carrier protein (rACP), a small acidic protein whose secondary and tertiary structure can be manipulated by pH, fatty acylation, and site-directed mutagenesis. Circular dichroism and intrinsic fluorescence demonstrated that apo-rACP adopts a folded helical conformation in aqueous solution below pH 6 or in 50% acetonitrile/0.1% formic acid, but is unfolded at neutral and basic pH values. A rACP mutant, in which seven conserved acidic residues were replaced with their corresponding neutral amides, was folded over the entire pH range of 5 to 9. However, under the same solvent conditions, both wild type and mutant ACPs exhibited similar CSDs (6(+)-9(+) species) at all pH values. Covalent attachment of myristic acid to the phosphopantetheine prosthetic group of rACP, which is known to stabilize a folded conformation in solution, also had little influence on its CSD in either positive or negative ion modes. Overall, our results are consistent with ACP as a "natively unfolded" protein in a dynamic conformational equilibrium, which allows access to (de)protonation events during the electrospray process.     

Modification of <Emphasis Type="Italic">Cytochrome c</Emphasis> by 4-hydroxy- 2-nonenal: Evidence for histidine,lysine, and arginine-aldehyde adducts

4-Hydroxy-2-nonenal (4HNE), a major secondary product of lipid peroxidation, has been associated with a number of disease states involving oxidative stress. Despite the recognized importance of post-translational modification of proteins by products such as 4HNE, little is known of the modification of cytochrome c by this reagent and its analysis by mass spectrometry. The purpose of this study was to investigate the chemical interaction of 4HNE and cytochrome c, a protein essential to cellular respiration, under in vitro conditions. Isoelectric focusing of native and 4HNE-modified cytochrome c using immobilized pH gradient (IpG) strips showed a decrease in the pI of the 4HNE-modified protein suggesting modification of charged amino acids. Reaction of 4HNE with cytochrome c resulted in increases in molecular weight consistent with the addition of four 4HNE residues as determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS). Samples of both native and 4HNE-modified cytochrome c were enzymatically digested and subjected to peptide mass fingerprinting using MALDI-TOF MS. Analysis of these samples using LC-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) provided sequence information that was used to determine specific residues to which the aldehyde adducted. Taken together, the data indicated that H33, K87, and R38 were modified by 4HNE. Mapping these results onto the X-ray crystal structure of native cytochrome c suggest that 4HNE adduction to cytochrome c could have significant effects on tertiary structure, electron transport, and ultimately, mitochondrial dysfunction.