Structure and Dynamics of a Protein–Surfactant Assembly Studied by

Aug 12, 2015 - The structure and dynamics of a protein–surfactant assembly studied by ion-mobility mass spectrometry (IMS) and vacuum molecular dyna...
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Structure and Dynamics of a Protein−Surfactant Assembly Studied by Ion-Mobility Mass Spectrometry and Molecular Dynamics Simulations Antoni J. Borysik* Department of Chemistry, King’s College London, Britannia House, London SE1 1DB, United Kingdom S Supporting Information *

ABSTRACT: The structure and dynamics of a protein− surfactant assembly studied by ion-mobility mass spectrometry (IMS) and vacuum molecular dynamics (MD) simulations is reported. Direct evidence is provided for the ability of the surfactant dodecyl-β-D-maltoside (DDM) to prevent chargeinduced unfolding of the membrane protein (PagP) in the gasphase. Restraints obtained by IMS are used to map the surfactant positions onto the protein surface. Surfactants occupying more exposed positions at the apexes of the β-barrel structure are most in-line with the experimental observations. MD simulations provide additional evidence for this assembly organization through surfactant inversion and migration on the protein structure in the absence of solvent. Surfactant migration entails a net shift from apolar membrane spanning regions to more polar regions of the protein structure with the DDM molecule remaining attached to the protein via headgroup interactions. These data provide evidence for the role of protein-DDM headgroup interactions in stabilizing membrane protein structure from gas-phase unfolding.

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ing which amphiphiles are best suited to the study of membrane proteins by MS must be determined experimentally as the behavior of these species and how they bind to and modulate protein structure cannot currently be predicted from what is known in solution.11,12 Experimental strategies to determine these effects typically rely on comparative approaches that rank different amphiphiles according to their ability to maintain compact protein structures after surfactant removal. These approaches have provided crucial insights into the role of amphipols in stabilizing gas-phase membrane proteins and have underpinned the development of surfactant screening protocols.13−15 However, from these comparative methods it is difficult to ascertain why certain amphiphiles work best for gas-phase studies. An alternative approach to address these questions is to characterize gas-phase membrane proteins under gentler conditions preserving the protein−surfactant interactions and studying the structure and dynamics of the assemblies. The disassembly pathways of these species can be followed, and the effect of surfactants on protein structure can be visualized directly without inferral to a reference state. The organization of surfactants mapped onto the protein structure can also be investigated to reveal important clues as to how these molecules stabilize protein structures in the solvent-free environment. Here is reported the structure and dynamics of a surfactantmembrane protein assembly studied by Ion-Mobility Mass

erhaps the most exciting recent breakthrough in gas-phase structural biology has been the successful extension of these methods to the study of membrane proteins.1 Since these pioneering experiments interest in the use of gas-phase methods to study membrane proteins has surged due to the potential they hold in paving new inroads into understanding these important yet challenging proteins. Free of solvent effects the structure and dynamics of membrane proteins can be studied unhindered.2,3 Crucial information regarding the binding of these proteins to important drug targets or lipids can also be accessed more readily.4,5 The gas-phase characterization of functional membrane proteins typically involves large adjustments to the instrumental collision voltages to values which would be deemed unfavorably high for the study of soluble proteins.6 These settings are required to liberate membrane proteins from surfactant micelles or other membrane mimics, such as nanodiscs or bicelles.7 Gasphase “desolvation” reduces ambiguities brought about from peak broadening due to the presence of multiple surfactant adducts bound to the protein. It is remarkable that membrane protein complexes can be subjected to these conditions and maintain their native stoichiometry and compact structure. Membrane proteins typically have reduced ionization states relative to soluble proteins, and charge reduction can contribute to the apparent stability of membrane proteins in the gasphase.8 The stabilizing effect of small molecules is well-known, and surfactants have also been shown to contribute directly to protein stability in the absence of solvent.9,10 There is emerging interest in the molecular mechanisms of characterizing membrane proteins in the gas-phase. Determin© XXXX American Chemical Society

Received: June 9, 2015 Accepted: August 12, 2015

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DOI: 10.1021/acs.analchem.5b02172 Anal. Chem. XXXX, XXX, XXX−XXX

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with helium-based Ω of known standards of ubiquitin (SigmaAldrich, U253) cytochrome c (Sigma, Aldrich 30396) and horse heart myoglobin (Sigma-Aldrich, 30396). Drift times were obtained from peak apexes after fitting the arrival time distributions to a minimal number of Gaussian functions. Postprocessing of data was performed with minimal smoothing and without subtraction. Instrumental settings were Capillary (1.3 kV), Cone (50 V), Trap bias (30 V), Transfer CE (10 V), IMS wave height (30 V), and IMS wave velocity (300 V). The Trap CE was varied between 10 and 100 V for the respective low and high energy conditions referred to in the main text. Argon, nitrogen, and helium gas-flow rates were 8, 180, and 90 mL/min, respectively. Under these settings the Ω and populations sizes of the protein−surfactant assemblies obtained by TWIMS were highly reproducible (Figure S1). All mobiligrams are represented by a square root 2D map intensity scale. Modeling and Molecular Dynamics Simulations of PagP. Missing loops and residues from the crystal structure of PagP (1THQ) were built using Modeller to generate 100 structures.17 The structure with the lowest zDOPE score (−1.06) was selected and studied further by molecular dynamics (MD) simulations. All MD simulations were performed using the OPLS/AA force field implemented within GROMACS 4.6.7.18 For vacuum simulations of the surfactantfree form of PagP various charge configurations were initially permuted for the 6+ and 8+ ions of the protein by neutralization of acidic residues assuming initial protonation states of all charged residues at pH 7.0; histidine residues were considered neutral. For the 6+ charge state production MD simulations were performed on the charge isomer with the lowest Coulomb energy following steepest descent energy minimization. Simulations followed previously reported protocols.19 Neither periodicity nor cut-offs were used during the calculations. A small integration time step of 1 fs was used to ensure energy conservation, and bonds to H atoms were constrained using the LINCS algorithm.20 Unfolded PagP structures were generated by initially heating a range of different charge isomers for the 8+ ion to 1000 K. Partially unfolded states were taken along these trajectories, the charge was redistributed, and the new structures were subjected to simulated annealing involving successive heating of the structures to 800 K followed by rapid cooling to 300 K allowing the protein to explore various partially unfolded states; theoretical Ω were obtained with Impact.21 MD Simulations of PagP:DDM Assemblies. The embedding of PagP in a DDM micelle was initially carried out by randomly packing 100 DDM molecules around a charge neutral protein with Packmol.22 This structure was then solvated and energy minimized to eliminate unfavorable contacts and steric overlaps followed by extensive solvent equilibration. DDM topology files were obtained from lipidbook.23 Production MD simulations were performed at 300 K for 100 ns after which time the surfactants had formed an ordered micelle around the protein. Vacuum simulations of the protein embedded in a 100 DDM micelle were performed as per the surfactant-free protein at 300 K. Micelle inversion was brought about by gradual heating of the assembly to 600 K from 0 ns−12 ns followed by continued simulation at 600 K. Micelle Rg plots were prepared with separate index files for the DDM headgroup and alkyl chain carbon atoms. Mapping of the overall positions of the DDM surfactants on the surface of PagP was achieved by comparing the

Spectrometry (IMS) and Molecular Dynamics (MD) simulations. At high collision energies the protein present in the surfactant-free form as a mixture of collapsed and charge unfolded states. However, a third transiently populated nativelike structure is observed at reduced energy with the protein assembled with the surfactant dodecyl-β-D-maltoside (DDM). These experiments directly capture for the first time the ability of protein-bound surfactants to shield against charge-induced unfolding of a gas-phase membrane protein. Tandem-MS (MSMS) IMS experiments reveal that protection against unfolding is afforded to those populations that remain surfactant assembled. This contrasts with the ability of surfactants to protect against conformational collapse in which the release of these species is required. Vacuum MD simulations of PagP map the most significant structural changes to regions outside of the membrane spanning β-barrel of the protein; which remains largely intact over a range of different temperatures. Protein−surfactant assemblies were then studied in silico to understand the orientation and positions of surfactants on the protein surface. Evidence is provided for the inversion of DDM micelles on protein structures with a concomitant increase in headgroup mediated protein−surfactant hydrogen bonding. Clues relating to the mechanism by which surfactants protect membrane proteins from gas-phase unfolding are provided by MD simulations. Surfactant displacement from apolar regions to thermally labile polar protein surfaces at apexes of the membrane spanning β-barrel is observed and validated by IMS. These observations provide a rationale for chemical surfactant effects that shield membrane proteins from gas-phase unfolding. Surfactant migration from stable membrane spanning regions to unstable polar loops at the β-barrel apexes and α-helix of the protein position the surfactants to provide additional tethers to prevent gas-phase unfolding. The consequences of these findings are discussed within the context of characterizing membrane protein by MS.

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EXPERIMENTAL SECTION Protein Expression, Purification, and Refolding. Wildtype PagP was expressed with a C-terminal 6His tag in E. coli BL21(DE3) cells from the plasmid pETCrcAHΔS (kind gift from Russell Bishop, McMaster University) and then resolubilized from inclusion bodies into denaturing buffer (6 M Gdn-HCl and 10 mM Tris-HCl pH 8.0) as described previously.10,16 Protein was refolded by the drop dilution method into a refolding buffer of 0.5% LDAO (N,Ndimethyldodecylamine N-oxide) 10 mM Tris-HCL pH 8.0 as described previously.10,16 Correct folding of PagP was determined by far UV circular dichroisom and a functional activity assay involving turnover of the lipid analogue pNPP (4nitropheynyl palmitate Sigma, UK) as described previously.10 Ion Mobility Mass Spectrometry. Mass spectra of PagP were obtained in positive ion mode on a second Generation Synapt HDMS (Waters Corp. Wilmslow, UK) instrument fitted with a nanoflow electrospray ionization source (nano-ES). Refolded protein was thawed and buffer exchanged at least once into 200 mM ammonium acetate and 0.02% DDM using a P6̅ Micro Bio-Spin column (Bio-Rad, Hercules, CA). The final protein concentration was ∼10 μM. Arrival time distributions were extracted from m/z peaks at half-height. Drift times were obtained in nitrogen by traveling wave ion mobility MS (TWIMS) as previously described.10 Ion drift times were converted to collision cross sections by instrument calibration B

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Analytical Chemistry experimental Ω of the assemblies to theoretical values extracted from MD trajectories. For these experiments the low mass of PagP (∼20 kDa) is particularly useful as the mass of a single DDM molecule (510 Da) contributes significantly to the overall size of the assembly. Surfactant effects should therefore be magnified with this protein, and the positions of bound DDM molecules can be mapped more definitively. PagP:7DDM assemblies were initially prepared using Packmol with the surfactants positioned randomly around the central membrane spanning region of the protein. The assemblies were then energy minimized as per the surfactant free protein, prior to a short (250 ps) production MD simulation at 300 K to ensure the binding of the surfactants to the β-barrel of the protein. During the binding process position restraints were applied to the protein to prevent its collapse prior to further MD. After the binding of DDM to the protein was assured, a 20 ns vacuum MD simulation was performed without protein position restraints to allow the collapse of the structure. Surfactant positions were mapped using the collapsed state of PagP as this conformation should be the least dynamic of the various protein populations. Accordingly, surfactant contributions to assembly Ω of this state can be interpreted by their positional effects rather than any effect on protein structure. The protein was maintained charge neutral to minimize any influence of surfactant migration depending on charge positions. The collapse of 6+ and charge neutral and PagP species were identical. The presence of 7DDM on the surface of PagP provided no steric hindrance to the collapse of the protein (Figure S2). Orientations of DDM molecules on the collapsed protein structures were explored by gradual heating of the assemblies to 600 K followed by continued simulation at this temperature to increase conformational sampling of the surfactant. During this process backbone position restraints were applied to the protein α-carbons to prevent thermal disruption. Simulation temperatures in excess of 600 K were avoided as these were found to promote spontaneous detachment of surfactants from the protein. During the simulations the protein was found to undergo a mild (∼5%) structural expansion regardless of the use of backbone restraints. Structural expansion was a consequence of the temperature gradient which brought about movement of the protein side chains from their initial positions where they were collapsed onto the protein surface. All atom restraints could not be applied to the protein as the surfactants failed to migrate in these simulations, suggesting the need for some flexibility in the protein side chains to assist in surfactant displacement. Reporting the contribution of DDM to the overall size of the assemblies during the MD trajectories was achieved as follows. Assembly structures were extracted from the trajectories at intervals of 10 ps to obtain 1500 structures. A second pool of 1500 structures was then prepared from these assemblies by extraction of the DDM coordinates. ΔΩ were obtained by subtraction of the detergent-free Ω from those obtained in the surfactant assembled state for each of the 1500 models. In this way any protein expansion effects during heating were corrected (Figure S3). The theoretical ΔΩ were then compared to the equivalent experimental metrics obtained by IMS.

molecular mechanisms of studying membrane proteins by MS.10,13 PagP was overexpressed and purified as previously described (Experimental Section). Mass spectra were obtained in DDM (0.02% CMC) and in 200 mM ammonium acetate on a Waters second Generation Synapt HDMS as described previously (Experimental Section). At high energy (100 V Trap) PagP presents in the gas-phase in the surfactant-free form with each ion displaying largely monodisperse structures with compact geometries at low charge (Ω = 17.4 to 18.4 nm2 for the 7+ to 5+ ions) and more extended structures (Ω > 22.0 nm2) for charge states ≥8+ (Figures 1a−1b). To obtain

Figure 1. Gas-phase structures of PagP: Mass spectra and IMS mobiligram of PagP obtained on a Waters second Generation Synapt HDMS. Spectra were obtained at high (a−b) or low (c−d) energy conditions (Methods). Ω (nm2) of selected species are given in italics. Structures of respective species are shown along with the conformation of the native-like species (Ω = 19.0 nm2, part d insert). Several peaks originating from “empty” surfactant assemblies are observed as indicated.

respective structures for these species the protein was studied by vacuum MD simulation. Missing regions (15%) from the crystal structure of PagP (1THQ) were rebuilt using Modeler and the structure with the lowest energy (zDOPE-score) used for production MD (Experimental Section). A range of different charge states and charge configurations were prepared and production simulations performed using the OPLS/AA force field implemented with GROMACS 4.5.6. (Experimental Section). Free of solvent PagP collapses spontaneously with a corresponding reduction in Ω of ∼8% from 19.0 nm2 to 17.7 nm2 (Figures S4a and S1f). The backbone RMSD of the collapsed protein relative to the starting structure shows that collapse occurs most significantly in the flexible loop regions inbetween the structured β-barrel of the protein as proposed previously (Figure S4b).10 In silico unfolding of PagP is characterized by large disruptions of the N-terminal α-helix and distortions to the intervening loops, particularly the larger A/B loop of the protein (Figure S4b). The β-barrel of PagP is relatively stable in vacuum conditions, and the positions of the respective atoms remain mostly intact throughout the MD simulations. The Ω of the structures obtained by MD indicate that PagP presents as a mixture of collapsed and charge unfolded states in the gas-phase under high energy (surfactantstripping) conditions. No structures with Ω equivalent to values

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RESULTS Gas-Phase Structures of PagP. PagP is a bacterial outer membrane protein involved in the maintenance of lipid-A. It has been used previously as a model protein to characterize the C

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Figure 2. Following the disassembly of charge-unfolded species by MS: Stacked ATDs for the 6+ (a) and 8+ (b) PagP:DDM assemblies. c−d MSMS spectra and associated mobiligram for the 6+ PagP:6DDM assembly. e−f MSMS spectra and associated mobiligram for the 8+ PagP:7DDM assembly, example assembly structures are shown. The precursor and product ions in all MSMS spectra are color coded red and blue, respectively. g−h stacked ATDs for the 6+ (g) and 8+ (h) assemblies obtained from the respective mobiligrams obtained by MSMS. Collapsed, native-like, and unfolded populations are color coded blue, green, and red, respectively. A minor contribution to the assembly ATD from “empty” DDM micelles is color coded gray (asterisk).

expected for the native-like protein (Ω = 19.0 nm2) are observed. Mass spectra were then obtained under gentler instrumental settings more akin to those utilized for the characterization of soluble proteins by native-MS (Experimental Section). Under these conditions the protein−surfactant interactions are maintained as evidenced by the additional adduct peaks visible in the mass spectrum of PagP (Figure 1c). An additional feature is also apparent in the IMS contour plot of the protein with a Ω equivalent to that expected for the native-like structure of the protein (Figure 1d). For the collapsed ions the additional species correspond to the slower component of PagP as previously described. Conversely, for the charge unfolded states the additional species represent more compact, i.e. faster, components in the arrival time distribution (ATD) of the protein with a Ω corresponding to that of the crystal structure. IMS studies of PagP-DDM assemblies provide direct evidence of the ability of DDM to preserve native-like structures protecting the protein from conformational collapse and charge-induced unfolding in the gas-phase. Following the Disassembly of Charge-Unfolded Species by MSMS. Extraction of the ATDs of the 6+ and 8+ ions of the PagP:DDM assemblies underscores the involvement of DDM in protecting PagP due to the clear dose-dependence between DDM and the survival of native-like conformations (Figures 2a−2b). Previous experiments established a detergent release mechanism that could rescue PagP from conformational collapse.10 These mechanistic experiments involved the isolation of unique assemblies by MSMS, with the

resulting MSMS spectra and corresponding mobiligrams reporting on structural changes in the isolated assemblies as they break apart in the gas-phase. The mechanisms outlined previously for the protection of PagP from collapse and chargeinduced unfolding were then compared. Assemblies were isolated for both the 6+ and 8+ ionization states of the protein and MSMS spectra of each species obtained (Experimental Section). The MSMS spectra span a wide range of assembly sizes as the complexes disassemble in the gas-phase (Experimental Section). Each component is clearly visible in the mobiligrams and provides important mechanistic information on the consequences of surfactant release or retention in the gas-phase (Figures 2c−2f). Akin to previous studies of PagP the rescue of native-like structures from gas-phase collapse is commensurate with the release of bound surfactants from the initially isolated assemblies (Figure 2g). The disassembly of the charge unfolded state displays an inverted mechanism relative to the collapsed conformation. Assemblies that remain surfactant associated following initial selection by MSMS (7DDM) are characterized by large populations (∼60%) of native-like structures. In contrast a clear shift toward charge unfolded states (∼90%) is evident for those populations that release bound surfactants following initial selection (Figure 2h). These data clearly show that surfactants must remain bound to membrane proteins in order for them to exert protection from charge induced unfolding in the gas-phase. IMS Predicts Exposed Surfactant Orientations in the Gas-Phase. The stacked ATDs of the protein−surfactant D

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(DPC). These findings were more recently expanded upon in a comprehensive MD study by Rouse et al. which described different inversion propensities for DDM and DPC.24 DDM micelles largely maintained solution orientations due to extensive hydrogen bonding networks throughout their maltoside head-groups. The ability of DDM micelles to maintain solution configurations conferred additional protection to the membrane protein over DPC micelles, which inverted readily exposing membrane-spanning protein regions to harmful vacuum conditions. MD studies provide little evidence for any protective role of the DDM head groups as these moieties make few contacts with the protein during the vacuum simulations. However, the effect of higher energy conditions on surfactant orientation has not been explored. These conditions may better reflect those expected by experiment particularly during surfactant disruption. Higher temperatures will also permit greater conformational sampling of the surfactants which may be required due to short time scales of the simulations and any possible overrepresentation of nonbonded interactions in vacuum MD simulations.25,26 To address these questions a PagP:DDM assembly comprising of 100 DDM molecules was prepared and equilibrated in water to enable micelle formation around the protein. The assembly was then desolvated and studied in vacuum at ambient temperature (Experimental Section, Figure S5). The relative positions of the DDM head-groups and alkyl chain moieties were followed by calculation of the radius of gyration of the respective carbon atoms during the trajectory. Following initial contraction of the assembly the relative positions of the DDM head-groups and alkyl chains run parallel to each other with no significant inversion of these moieties observed (Figure 4a). Overall these results agree with those observed previously for OmpA in DDM with regard to the general preservation of the micellar structure around the protein in vacuum conditions. The assembly was then heated gradually to 600 K to characterize temperature effects on the overall geometry of the DDM micelle. Temperature increases were accompanied by a spontaneous inversion of the surfactants within the micelle (Figure 4b). The assembly adopts an altered configuration in which the surfactant headgroups contact the protein and the alkyl moieties point outward to the gas-phase. DDM-DDM hydrogen bond contacts are replaced for those involving the protein as the surfactants invert (Figure S6). No surfactant ejection is observed during the simulations at higher temperature suggesting that surfactant loss transpires after the inversion of these species in gas-phase. An assembly of PagP and 7 DDM molecules was then prepared. Surfactants were initially positioned to neighbor the membrane spanning regions of the protein and production MD simulations performed at ambient temperature to facilitate surfactant binding and equilibration. DDM provided no discernible steric hindrance to the conformational collapse of PagP (Experimental Section). A gradual temperature gradient was then applied with the protein α-carbons restrained to permit conformational sampling of the surfactants without disruption of the overall protein structure. During the MD simulation the surfactants are shown to explore the protein surface to eventually reside at the polar apexes of the structure (Figure S7). Ultimately the surfactants remain attached to the protein by interactions mediated by their maltoside head groups. The overall geometry of the assemblies is altered as the surfactants become exposed on the protein surface. The ΔΩ was then obtained for structures along the assembly trajectories

assemblies show a clear shift toward larger Ω with an increasing number of bound surfactants (Figure 2). The change in Ω (ΔΩ) reflects the contribution of DDM to the overall size of each assembly. This parameter provides important clues regarding the location of surfactants bound to the protein surface and the mechanism by which these species protect membrane proteins from unfolding in the gas-phase. For the 6+ ions, the Ω of the collapsed species increases from 17.7 nm2 to 20.1 nm2 from the surfactant-free to the 7DDM assembled state. For the native-like configurations of PagP the Ω increases from 19.2 nm2 to 22.1 nm2 for the equivalent increase in bound surfactant. Accordingly, the ΔΩ (from surfactant-free to the addition of 7DDM) is 2.4 nm2 and 2.9 nm2 for the collapsed and native-like species, respectively (Figure 3a).

Figure 3. Characterizing the assembly ΔΩ: a Overlay of the ATDs of the surfactant-free (0DDM) and 7DDM assemblies of the 6+ charge state. Collapsed and native-like structures are color coded blue and green, respectively. The ΔΩ between the assemblies is indicated for each state. b Modeled PagP:7DDM assemblies for the collapsed configuration. Structures were initially prepared with Packmol following vacuum simulation at 300 K (Experimental Section). Burying of DDM within the structure of PagP is evident on inspection of the structures. The gray population evident in (a) originates from a free DDM assembly extracted on the same m/z coordinate.

To understand the relationship between the experimental ΔΩ values and the surfactant positions a PagP:7DDM assembly was prepared and studied in vacuum by MD (Experimental Section). Surfactants were initially positioned around the apolar β-barrel membrane spanning region of the protein as expected in solution. The ΔΩ obtained for the assemblies were in general 3−4-fold lower than observed by experiment (0.7 nm2). Inspection of the model assemblies revealed the surfactants occupying crevices within the membrane spanning region of the protein structure. In this configuration the surfactants are concealed such that they contribute very little change in the apparent size of the structure (Figure 3b). These experiments suggest that the surfactants adopt more exposed configurations on the protein in the gas-phase, potentially migrating from obscured membrane spanning regions where interactions would be more favorable in solvent to more exposed polar protein surfaces, rich in thermally labile loops at the apexes of the βbarrel of the protein. DDM Is Characterized by Inversion and Migration in the Gas-Phase. Reversed micelle orientations, surrounding membrane proteins in the gas-phase, were first proposed by Friemann and co-workers from in silico studies of OmpA encapsulated in the surfactant n-dodecylphosphocholine E

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Figure 5. Change in Ω during surfactant migration: Plot of ΔΩ during the vacuum MD simulation of a PagP:7DDM assembly. The ΔΩ observed by IMS for the collapsed 6+ ion of PagP is shown (dashed line). Several assembly structures are also shown at various time-points along the trajectory.

that surfactants reside in loops and helices prior to ejection from the protein.

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DISCUSSION The conformations of PagP:DDM assemblies have been studied without solvent by IMS and MD simulations. Direct evidence for the stabilizing effect of DDM on the gas-phase unfolding of PagP has been provided. MSMS-IMS experiments have shown that continued protein−surfactant interactions are required to resist against gas-phase protein unfolding. Evidence for micellar inversion is provided by vacuum MD simulations where thermal heating disrupts intramolecular DDM structure to enabling surfactant inversion resulting in increased proteinDDM headgroup interactions. Surfactants buried in protein crevices along membrane spanning regions provide insufficient contributions to the overall assembly size to satisfy the experimental ΔΩ. These observations support a process involving surfactant migration whereby these species become more exposed along the protein structure. The migration of DDM from apolar membrane spanning regions to polar protein regions is replicated by vacuum MD simulation, and this process transpires prior to surfactant release from the protein. The surfactant alkyl chains potentially collapse back onto the protein surface, but a redistribution of surfactant mass to the apexes of the proteins β-barrel structure must be required to increase the overall shape anisotropy of the assemblies. The organization of protein−surfactant assemblies in gas-phase are unlikely to represent the organization of these species anticipated in solution. These data implicate a role for the maltoside head-groups and hydrogen bonding on stabilizing membrane protein structure from unfolding in the gas-phase. Surfactants have the ability to modulate and preserve the structures of membrane proteins in the gas-phase. It is interesting that surfactants maintain this property in this environment given the fundamentally different properties of surfactant assemblies in the absence of solvent.11,12,27 The protective property of surfactants is seemingly maintained across different environments due to the adaptability of the amphipathic nature of these species. Membrane spanning protein surfaces that are intolerant to water shelter under

Figure 4. Thermally induced inversion of a gas-phase DDM micelle: Plots of the relative positions (Rg) of the DDM headgroup and alkyl chain carbon atoms during the course of MD simulation in vacuum at 300 K (a) and 300−600 K (from 0−12 ns) (b). Representative structures at 16 ns are shown with surfactants removed to allow visualization of PagP.

(Experimental Section). The initial ΔΩ of the assemblies increases significantly over the course of the simulations. Surfactant displacement is commensurate with an ∼4-fold increase in ΔΩ to reach values compatible to those observed by IMS (Figure 5). For structures that satisfy the experimental Ω the DDM molecules have undergone a process of migration on the protein structure. Surfactants vacate the more stable apolar β-barrel of the protein to bind to the more thermally labile polar structure at the barrel apexes and α-helix. As observed previously for PagP embedded in a DDM micelle surfactant displacement transpires prior to surfactant release suggesting F

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(8) Mehmood, S.; Marcoux, J.; Hopper, J. T.; Allison, T. M.; Liko, I.; Borysik, A. J.; Robinson, C. V. J. Am. Chem. Soc. 2014, 136, 17010. (9) Han, L.; Hyung, S. J.; Ruotolo, B. T. Faraday Discuss. 2013, 160, 371. (10) Borysik, A. J.; Hewitt, D. J.; Robinson, C. V. J. Am. Chem. Soc. 2013, 135, 6078. (11) Borysik, A. J.; Robinson, C. V. Phys. Chem. Chem. Phys. 2012, 14, 14439. (12) Borysik, A. J.; Robinson, C. V. Langmuir 2012, 28, 7160. (13) Leney, A. C.; McMorran, L. M.; Radford, S. E.; Ashcroft, A. E. Anal. Chem. 2012, 84, 9841. (14) Calabrese, A. N.; Watkinson, T. G.; Henderson, P. J.; Radford, S. E.; Ashcroft, A. E. Anal. Chem. 2015, 87, 1118. (15) Reading, E.; Liko, I.; Allison, T. M.; Benesch, J. L.; Laganowsky, A.; Robinson, C. V. Angew. Chem., Int. Ed. 2015, 54, 4577. (16) Khan, M. A.; Neale, C.; Michaux, C.; Pomes, R.; Prive, G. G.; Woody, R. W.; Bishop, R. E. Biochemistry 2007, 46, 4565. (17) Fiser, A.; Do, R. K.; Sali, A. Protein Sci. 2000, 9, 1753. (18) Van Der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. J. Comput. Chem. 2005, 26, 1701. (19) Hall, Z.; Politis, A.; Bush, M. F.; Smith, L. J.; Robinson, C. V. J. Am. Chem. Soc. 2012, 134, 3429. (20) Edberg, R.; Evans, D. J.; Morriss, G. P. J. Chem. Phys. 1986, 84, 6933. (21) Marklund, E. G.; Degiacomi, M. T.; Robinson, C. V.; Baldwin, A. J.; Benesch, J. L. Structure 2015, 23, 791. (22) Martinez, L.; Andrade, R.; Birgin, E. G.; Martinez, J. M. J. Comput. Chem. 2009, 30, 2157. (23) Domanski, J.; Stansfeld, P. J.; Sansom, M. S.; Beckstein, O. J. Membr. Biol. 2010, 236, 255. (24) Rouse, S. L.; Marcoux, J.; Robinson, C. V.; Sansom, M. S. Biophys. J. 2013, 105, 648. (25) Fraternali, F.; Van Gunsteren, W. F. J. Mol. Biol. 1996, 256, 939. (26) Leontyev, I. V.; Stuchebrukhov, A. A. J. Chem. Theory Comput. 2010, 6, 1498. (27) Longhi, G.; Fornili, S. L.; Turco Liveri, V. Phys. Chem. Chem. Phys. 2015, 17, 16512.

surfactant alkyl moieties in solution. In the gas-phase surfactants cling to the protein via headgroup interactions. Surfactant migration positions the maltoside head groups at polar protein surfaces that are more labile to gas-phase unfolding where they can provide tethers of structural support through hydrogen bonding. Presumably the displacement of surfactants from lipophilic membrane spanning regions to lyophobic surfaces is driven by a tendency of the surfactant head-groups to maximize their hydrogen bonding to the protein. An interesting property of DDM is its ability to capture transient native-like protein conformations and enable their visualization in the gas-phase. Surfactants may have exciting potential to enhance the study of proteins using gas-phase methods. However, surfactant “stickiness” remains an unfavorable property in the majority of experimental workflows. The need for additional thermal agitation to remove these sticky species and enable protein characterization typically has deleterious consequences on protein structure. Future directions may focus on increased resolution at lower energy. This may permit the characterization of gas-phase membrane proteins embedded in large lipid/surfactant environments where native structures/interactions of these proteins may be easier to capture prior to gas-phase rearrangement.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02172.

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Figures S1−S7 (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: 44(0)20 7848 7508. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Kind thanks are expressed to Dr. Erik Marklund for various MD bug fixes through this article and also to Drs. Erik Marklund, Zoe Hall, and Matteo Degiacomi and Prof. Carol Robinson for some useful discussions.

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REFERENCES

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DOI: 10.1021/acs.analchem.5b02172 Anal. Chem. XXXX, XXX, XXX−XXX