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Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins due to their presence of impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Herein, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium-labeled protein-lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein-lipid nanodisc─both empty and loaded with the ∼115 kDa transmembrane protein AcrB─proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO2-coated and TiO2 beads and describe how solution conditions can be optimized to maximize not only the performance of our online but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial lipid bilayers or even native cell membranes.

Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system fromEscherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αβ domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.

Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins, due to their presence impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Here, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium labeled protein-lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein lipid nanodisc – both empty and loaded with the ~115 kDa transmembrane protein AcrB – proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and with minimal protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO2-coated and TiO2 beads, and describe how solution conditions can be optimized to maximize the performance of our online, but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial lipid bilayers or even cell membranes.

Multidrug efflux pumps are ubiquitous across both eukaryotes and prokaryotes, and have major implications in antimicrobial and multidrug resistance. They reside within cellular membranes and have proven difficult to study owing to their hydrophobic character and relationship with their compositionally complex lipid environment. Advances in structural mass spectrometry (MS) techniques have made it possible to study these systems to elucidate critical information on their structure–function relationships. For example, MS techniques can report on protein structural dynamics, stoichiometry, connectivity, solvent accessibility, and binding interactions with ligands, lipids, and other proteins. This information proving powerful when used in conjunction with complementary structural biology methods and molecular dynamics (MD) simulations. In the present review, aimed at those not experts in MS techniques, we report on the current uses of MS in studying multidrug efflux systems, practical considerations to consider, and the future direction of the field. In the first section, we highlight the importance of studying multidrug efflux proteins, and introduce a range of different MS techniques and explain what information they yield. In the second section, we review recent studies that have utilised MS techniques to study and characterise a range of different multidrug efflux systems.

SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic surface. Spikes of variants of concern (VOCs) acquired amino acid changes linked to increased virulence and immune evasion. Here, using HDX-MS, we identified changes in spike dynamics that we associate with the transition from closed to open conformation, to ACE2 binding, and to specific mutations in VOCs. We show that the RBD-associated subdomain plays a role in spike opening, whereas the NTD acts as a hotspot of conformational divergence of VOC spikes driving immune evasion. Alpha, beta and delta spikes assume predominantly open conformations and ACE2 binding increases the dynamics of their core helices, priming spikes for fusion. Conversely, substitutions in omicron spike lead to predominantly closed conformations, presumably enabling it to escape antibodies. At the same time, its core helices show characteristics of being pre-primed for fusion even in the absence of ACE2. These data inform on SARS-CoV-2 evolution and omicron emergence.

In order to comprehend the molecular basis of transmembrane protein biogenesis, methods are required that are capable of investigating the co-translational folding of these hydrophobic proteins. Equally, in artificial cell studies, controllable methods are desirable for in situ synthesis of membrane proteins that then direct reactions in the synthetic cell membrane. Here we describe a method that exploits cell-free expression systems and tunable membrane mimetics to facilitate co-translational studies. Alteration of the lipid bilayer composition improves the efficiency of the folding system. The approach also enables membrane transport proteins to be made and inserted into artificial cell platforms such as droplet interface bilayers. Importantly, this gives a new facet to the droplet networks by enabling specific transport of molecules across the synthetic bilayer against a concentration gradient. This method also includes a protocol to pause and restart translation of membrane proteins at specified positions during their co-translational folding. This stop-start strategy provides an avenue to investigate whether the proteins fold in sequence order, or if the correct fold of N-terminal regions is reliant on the synthesis of downstream residues.

Clinically relevant multidrug-resistant bacteria often arise due to overproduction of membrane-embedded efflux proteins that are capable of pumping antibiotics out of the bacterial cell before the drugs can exert their intended toxic effect. The Escherichia coli membrane protein AcrB is the archetypal protein utilized for bacterial efflux study because it can extrude a diverse range of antibiotic substrates and has close homologues in many Gram-negative pathogens. Three AcrB subunits, each of which contains 12 transmembrane (TM) helices, are known to trimerize to form the minimal functional unit, stabilized noncovalently by helix-helix interactions between TM1 and TM8. To inhibit the efflux activity of AcrB, we have rationally designed synthetic peptides aimed at destabilizing the AcrB trimerization interface by outcompeting the subunit interaction sites within the membrane. Here we report that peptides mimicking TM1 or TM8, with flanking N-terminal peptoid tags, and C-terminal lysine tags that aid in directing the peptides to their membrane-embedded target, decrease the AcrB-mediated efflux of the fluorescent substrate Nile red and potentiate the effect of the antimicrobials chloramphenicol and ethidium bromide. To further characterize the motif encompassing the interaction between TM1 and TM8, we used Forster resonance energy transfer to demonstrate dimerization. Using the TM1 and TM8 peptides, in conjunction with several selected mutant peptides, we highlight residues that may increase the potency and specificity of the peptide drug candidates. In targeting membrane-embedded protein-protein interactions, this work represents a novel approach to AcrB inhibition and, more broadly, a potential route to a new category of efflux pump inhibitors.

Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snapshots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture in vivo-generated membrane protein RNCs within their native lipid compositions. We reveal that a polytopic membrane protein can be successfully stalled at various stages during its synthesis and the resulting RNC extracted within either detergent micelles or diisobutylene–maleic acid co-polymer native nanodiscs. Our approaches offer tractable solutions for the structural and biophysical interrogation of nascent membrane proteins of specified lengths, as the elongating nascent chain emerges from the ribosome and inserts into its native lipid milieu.

Resistance–nodulation–division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics., AcrB is a prototypical resistance–nodulation–division (RND) bacterial transporter, conferring resistance to a variety of antibiotics. HDX-MS and other, complementary approaches offer insight into AcrB structural dynamics and suggest the molecular mechanisms underlying drug export and inhibition of this multidrug-resistance conferring pump.

Resistance-nodulation-division (RND) efflux pumps play a key role in inherent and evolved multidrug-resistance (MDR) in bacteria. AcrB is the prototypical member of the RND family and acts to recognise and export a wide range of chemically distinct molecules out of bacteria, conferring resistance to a variety of antibiotics. Although high resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown, preventing a complete mechanistic understanding of efflux and inhibition. Here, we determine these structural dynamics in the presence of AcrB substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular modelling, drug binding and bacterial susceptibility studies. We show that the well-studied efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within an MDR clinical isolate, AcrBG288D, modifies the plasticity of the transport pathway, which could explain its altered substrate specificity. Our results provide molecular insight into drug export and inhibition of a major MDR-conferring efflux pump and the important directive role of its dynamics.

Secondary transporters undergo structural rearrangements to catalyze substrate translocation across the cell membrane - yet how such conformational changes happen within a lipid environment remains poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS) with molecular dynamics (MD) simulations to understand how lipids regulate the conformational dynamics of secondary transporters at the molecular level. Using the homologous transporters XylE, LacY and GlpT from Escherichia coli as model systems, we discover that conserved networks of charged residues act as molecular switches that drive the conformational transition between different states. We reveal that these molecular switches are regulated by interactions with surrounding phospholipids and show that phosphatidylethanolamine interferes with the formation of the conserved networks and favors an inward-facing state. Overall, this work provides insights into the importance of lipids in shaping the conformational landscape of an important class of transporters., info:eu-repo/semantics/published

Mass spectrometry has emerged as an important structural biology tool for understanding membrane protein structure, function, and dynamics. Generally, structural mass spectrometry of membrane proteins has been performed on purified or reconstituted systems which lack the native lipid membrane and cellular environments. However, there has been progress in the use and adaptations of these methods for probing membrane proteins within increasingly more native contexts. In this Concept article the use and utility of structural mass spectrometry techniques for studying membrane proteins within native environments are highlighted.

Significance Outer-membrane porins are often considered as passive conduits of small molecules across lipid bilayers. Using native mass spectrometry experiments we identify a pH-sensitive lipid-binding mechanism of outer membrane porin F, which enables increased threading of a colicin-derived peptide through open channels. Supported by molecular dynamics simulations and channel recording experiments, we posit that this mechanism attenuates channel opening in response to changes in environmental conditions, specifically pH. These findings have important consequences for mass spectrometry experiments, wherein the role of charge is often overlooked, and they also could help provide understanding of antibiotics that gain access to Gram-negative bacteria through porin-mediated pathways., Strong interactions between lipids and proteins occur primarily through association of charged headgroups and amino acid side chains, rendering the protonation status of both partners important. Here we use native mass spectrometry to explore lipid binding as a function of charge of the outer membrane porin F (OmpF). We find that binding of anionic phosphatidylglycerol (POPG) or zwitterionic phosphatidylcholine (POPC) to OmpF is sensitive to electrospray polarity while the effects of charge are less pronounced for other proteins in outer or mitochondrial membranes: the ferripyoverdine receptor (FpvA) or the voltage-dependent anion channel (VDAC). Only marginal charge-induced differences were observed for inner membrane proteins: the ammonia channel (AmtB) or the mechanosensitive channel. To understand these different sensitivities, we performed an extensive bioinformatics analysis of membrane protein structures and found that OmpF, and to a lesser extent FpvA and VDAC, have atypically high local densities of basic and acidic residues in their lipid headgroup-binding regions. Coarse-grained molecular dynamics simulations, in mixed lipid bilayers, further implicate changes in charge by demonstrating preferential binding of anionic POPG over zwitterionic POPC to protonated OmpF, an effect not observed to the same extent for AmtB. Moreover, electrophysiology and mass-spectrometry–based ligand-binding experiments, at low pH, show that POPG can maintain OmpF channels in open conformations for extended time periods. Since the outer membrane is composed almost entirely of anionic lipopolysaccharide, with similar headgroup properties to POPG, such anionic lipid binding could prevent closure of OmpF channels, thereby increasing access of antibiotics that use porin-mediated pathways.

The interplay between membrane proteins and the lipids of the membrane is important for cellular function, however, tools enabling the interrogation of protein dynamics within native lipid environments are scarce and often invasive. We show that the styrene-maleic acid lipid particle (SMALP) technology can be coupled with hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate membrane protein conformational dynamics within native lipid bilayers. We demonstrate changes in accessibility and dynamics of the rhomboid protease GlpG, captured within three different native lipid compositions, and identify protein regions sensitive to changes in the native lipid environment. Our results illuminate the value of this approach for distinguishing the putative role(s) of the native lipid composition in modulating membrane protein conformational dynamics., info:eu-repo/semantics/published

Ion mobility-mass spectrometry (IM-MS) in combination with molecular modeling offers the potential for small molecule structural isomer identification by measurement of their gas phase collision cross sections (CCSs). Successful application of this approach to drug metabolite identification would facilitate resource reduction, including animal usage, and may benefit other areas of pharmaceutical structural characterization including impurity profiling and degradation chemistry. However, the conformational behavior of drug molecules and their metabolites in the gas phase is poorly understood. Here the gas phase conformational space of drug and drug-like molecules has been investigated as well as the influence of protonation and adduct formation on the conformations of drug metabolite structural isomers. The use of CCSs, measured from IM-MS and molecular modeling information, for the structural identification of drug metabolites has also been critically assessed. Detection of structural isomers of drug metabolites using IM-MS is demonstrated and, in addition, a molecular modeling approach has been developed offering rapid conformational searching and energy assessment of candidate structures which agree with experimental CCSs. Here it is illustrated that isomers must possess markedly dissimilar CCS values for structural differentiation, the existence and extent of CCS differences being ionization state and molecule dependent. The results present that IM-MS and molecular modeling can inform on the identity of drug metabolites and highlight the limitations of this approach in differentiating structural isomers.

Other than more widely used methods, the use of styrene maleic acid copolymers allows the direct extraction of membrane proteins from the lipid bilayer into SMALPs keeping it in its native lipid surrounding., Other than more widely used methods, the use of styrene maleic acid allows the direct extraction of membrane proteins from the lipid bilayer into SMALPs keeping it in its native lipid surrounding. Here we present the combined use of SMALPs and LILBID-MS, allowing determination of oligomeric states of membrane proteins of different functionality directly from the native nanodiscs.

Photoinitiated polymerization-induced self-assembly (photo-PISA) is an efficient approach to predictably prepare polymeric nanostructures with a wide range of morphologies. Given that this process can be performed at high concentrations and under mild reaction conditions, it has the potential to have significant industrial scope. However, given that the majority of industrial (and more specifically biotechnological) formulations contain mixtures of polymers and surfactants, the effect of such surfactants on the PISA process is an important consideration. Thus, to expand the scope of the methodology, the effect of small molecule surfactants on the PISA process, specifically for the preparation of unilamellar vesicles, was investigated. Similar to aqueous photo-PISA findings in the absence of surfactant molecules, the originally targeted vesicular morphology was retained in the presence of varying concentrations of non-ionic surfactants, while a diverse set of lower-order morphologies was observed for ionic surfactants. Interestingly, a critical micelle concentration (CMC)-dependent behavior was detected in the case of zwitterionic detergents. Additionally, tunable size and membrane thickness of vesicles were observed by using different types and concentration of surfactants. Based on these findings, a functional channel-forming membrane protein (OmpF porin), stabilized in aqueous media by surfactant molecules, was able to be directly inserted into the membrane of vesicles during photo-PISA. Our study demonstrates the potential of photo-PISA for the direct formation of protein–polymer complexes and highlights how this method could be used to design biomimicking polymer/surfactant nanoreactors.

The mechanosensitive channel of large conductance (MscL) acts as an emergency release valve for osmotic shock of bacteria preventing cell lysis. The large pore size, essential for function, requires the formation of oligomers with tetramers, pentamers, or hexamers observed depending on the species and experimental approach. We applied non-denaturing (native) mass spectrometry to five different homologs of MscL to determine the oligomeric state under more than 50 different experimental conditions elucidating lipid binding and subunit stoichiometry. We found equilibrium between pentameric and tetrameric species, which can be altered by detergent, disrupted by binding specific lipids, and perturbed by increasing temperature (37°C). We also established the presence of lipopolysaccharide bound to MscL and other membrane proteins expressed in Escherichia coli, revealing a potential source of heterogeneity. More generally, we highlight the use of mass spectrometry in probing membrane proteins under a variety of detergent-lipid environments relevant to structural biology.

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.

The HerA–NurA helicase–nuclease complex cooperates with Mre11 and Rad50 to coordinate the repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA–NurA. By combining hybrid mass spectrometry with cryo-EM, computational and biochemical data, we investigate the oligomeric formation of HerA and detail the mechanism of nucleotide binding to the HerA–NurA complex from thermophilic archaea. We reveal that ATP-free HerA and HerA-DNA complexes predominantly exist in solution as a heptamer and act as a DNA loading intermediate. The binding of either NurA or ATP stabilizes the hexameric HerA, indicating that HerA–NurA is activated by substrates and complex assembly. To examine the role of ATP in DNA translocation and processing, we investigated how nucleotides interact with the HerA–NurA. We show that while the hexameric HerA binds six nucleotides in an ‘all-or-none’ fashion, HerA–NurA harbors a highly coordinated pairwise binding mechanism and enables the translocation and processing of double-stranded DNA. Using molecular dynamics simulations, we reveal novel inter-residue interactions between the external ATP and the internal DNA binding sites. Overall, here we propose a stepwise assembly mechanism detailing the synergistic activation of HerA–NurA by ATP, which allows efficient processing of double-stranded DNA.

Natural proteins can be versatile building blocks for multimeric, self-assembling structures. Yet, creating protein-based assemblies with specific geometries and chemical properties remains challenging. Highly porous materials represent particularly interesting targets for designed assembly. Here, we utilize a strategy of fusing two natural protein oligomers using a continuous alpha-helical linker to design a novel protein that self assembles into a 750 kDa, 225 Å diameter, cube-shaped cage with large openings into a 130 Å diameter inner cavity. A crystal structure of the cage showed atomic-level agreement with the designed model, while electron microscopy, native mass spectrometry and small angle X-ray scattering revealed alternative assembly forms in solution. These studies show that accurate design of large porous assemblies with specific shapes is feasible, while further specificity improvements will probably require limiting flexibility to select against alternative forms. These results provide a foundation for the design of advanced materials with applications in bionanotechnology, nanomedicine and material sciences.

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.

Mass spectrometry (MS) of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes, where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here we describe a protocol for MS of membrane protein complexes. The protocol begins with the preparation of the membrane protein complex, enabling not only the direct assessment of stoichiometry, delipidation and quality of the target complex but also the evaluation of the purification strategy. A detailed list of compatible nonionic detergents is included, along with a protocol for screening detergents to find an optimal one for MS, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a quadrupole time-of-flight (Q-TOF) mass spectrometer after the introduction of complexes from gold-coated nanoflow capillaries.

Investigating the structures of membrane proteins and their interactions with lipids remains challenging for well-established biophysical techniques. In this thesis the use of mass spectrometry (MS) and ion mobility (IM) spectrometry were explored for the interrogation of membrane proteins, their stoichiometry, stability and interactions with lipids. The techniques used were also applied to the identification of drug metabolites. In the first two chapters reviews of both mass spectrometry methods, and membrane protein biogenesis and membrane protein-lipid interactions are presented. The first challenge for studying membrane proteins by MS was to optimise solution conditions. A detergent screening strategy was developed for this purpose (Chapter 3). The various detergent environments studied revealed dramatic differences in mass spectral quality permitting investigation of membrane protein-lipid interactions. Changes were observed in the electrospray charging of membrane proteins and trends were established from an extensive collection of membrane proteins ejected from a wide variety of detergent environments. The physicochemical principles behind the MS of membrane proteins were deduced and are presented (Chapter 4). The results of these experiments led to a deeper understanding of the ionisation processes and the influence of detergent micelles on both charge state and release mechanisms. Experiments from a range of different micelles also allowed the influence of charge and its effects on the preservation of native-like membrane protein conformations to be monitored by IM-MS. By resolving lipid-protein interactions, and by monitoring the effects of lipid binding on the unfolding of three diverse membrane protein complexes, substantial differences in the selectivity of membrane proteins for different lipids were revealed (Chapter 5). Interestingly lipids that stabilised membrane proteins in the gas-phase were found to induce modifications in structure or function thus providing an approach to assess direct lipid contributions, and to rank order lipids based on their ability to modulate membrane proteins. Using the MS approaches developed here also enabled study of the diversity of oligomeric states of the mechanosensitive channel of large conductance (MscL) (Chapter 6). Results revealed that the oligomeric state of MscL is sensitive to deletions in its C-terminal domain and to its detergent-lipid environment. Additionally, a case study with GlakoSmithKline (GSK) was undertaken using IM-MS technology but in this case applied to the identification of drug metabolites (Chapter 7). The results showed that IM-MS and molecular modelling could inform on the identity of different drug metabolites and highlights the potential of this approach in understanding the structure of various drug metabolites.

Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent K D values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.

The mixed donor N-heterocyclic carbene (NHC)/thione ligand precursors [1-(3-R-2H-imidazol-1-yl-2-thione)methyl-3-R-2H-imidazol-2-ium]X, [HCSR]X (R = methyl, benzyl; X = Br, I), have been utilized to prepare a range of silver and palladium complexes. The coordination of CSR to silver(I) salts has been explored, providing dimeric complexes of the type [AgX(CSR)]2 (where R = methyl, benzyl; X = Br, I). Structural characterization of [AgX(CSBn)]2 revealed a bidentate coordination mode for the mixed donor ligand and dinuclear structures where the silver centers are bridged by two bromido centers. Palladium complexes bearing one or two CSR ligands have additionally been prepared either directly, utilizing [Pd(OAc)2] as precursor, or via transmetalation strategies. The dicationic complexes [Pd(CSR)2][X]2 and neutral complexes [PdX2(CSR)] (where R = methyl, benzyl; X = Br, I, PF6) have been synthesized and fully characterized. The CSR ligand in the aforementioned complexes does not undergo transformation of the NHC unit to a urea function, which had been found to occur in the previously reported copper complexes. Palladium complexes containing both NHC/thione and bis-phosphine ligands were also prepared. Complexes of the type [Pd(CSMe)(L2)][X]2 and [PdX(CSMe)(L2)][X] (where L2 = dppe, dppp; X = Br, OAc, I, PF6) were obtained. The presence of the bis-phosphine appears to destabilize the coordination of the NHC/thione ligand and as a consequence leads to its elimination from the complex.

The effects of protein–ligand interactions on protein stability are typically monitored by a number of established solution-phase assays. Few translate readily to membrane proteins. We have developed an ion-mobility mass spectrometry approach, which discerns ligand binding to both soluble and membrane proteins directly via both changes in mass and ion mobility, and assesses the effects of these interactions on protein stability through measuring resistance to unfolding. Protein unfolding is induced through collisional activation, which causes changes in protein structure and consequently gas-phase mobility. This enables detailed characterization of the ligand-binding effects on the protein with unprecedented sensitivity. Here we describe the method and software required to extract from ion mobility data the parameters that enable a quantitative analysis of individual binding events. This methodology holds great promise for investigating biologically significant interactions between membrane proteins and both drugs and lipids that are recalcitrant to characterization by other means., Relatively few techniques can quantitatively measure the effect of ligands on membrane protein stability. Here the authors demonstrate the use of ion-mobility mass spectrometry to accurately measure and quantify ligand-induced protein stabilization in the gas phase.