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.

Ordered assembly of monomeric human ␤ 2 -microglobulin (␤ 2 m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions (pH <5.0 at 37°C), wild-type ␤ 2 m assembles spontaneously into fibrils with different morphologies. Under these conditions, ␤ 2 m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of ␤ 2 m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9؉ ions. By contrast, under more acidic conditions (pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10؉ and 11؉ ions. The data are reinforced by investigations on two variants of ␤ 2 m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid.␤ 2 -microglobulin (␤ 2 m) 1 is one of a number of proteins known to aggregate into insoluble amyloid deposits in vivo (1-3). These amyloid deposits have been linked to various human maladies such as Alzheimer's disease (4), Huntington's chorea (5), and senile systemic amyloidosis (6). However, for the majority of the ϳ20 known amyloid-related proteins, the structural mechanism of amyloid formation is still unclear, and, for several proteins, the role that different aggregated states play in the manifestation of the disease symptoms remains unresolved (7).␤ 2 m is a small (11,860-dalton) extracellular protein that forms the nonpolymorphic light chain component of the major histocompatibility class I complex. The physiological role of ␤ 2 m has been likened to a chaperone in that it is required for the heavy chain of the major histocompatibility class I complex to fold into a stable secreted entity (8, 9). ␤ 2 m is an all ␤-sheet protein, with a seven-stranded ␤-sandwich structure and a single disulfide bond linking Cys-25 and Cys-80 in the B and F strands (10) (Fig. 1). In healthy individuals, the serum concentration of circulating free ␤ 2 m is ϳ3 mg⅐liter Ϫ1 (11), and the protein is removed from the serum by renal catabolism (12). In individuals undergoing renal dialysis, the serum concentration of ␤ 2 m can rise to...

The study of intact soluble protein assemblies by means of mass spectrometry is providing invaluable contributions to structural biology and biochemistry. A recent breakthrough has enabled similar study of membrane protein complexes, following their release from detergent micelles in the gas phase. Careful optimization of mass spectrometry conditions, particularly with respect to energy regimes, is essential for maintaining compact folded states as detergent is removed. However, many of the saccharide detergents widely employed in structural biology can cause unfolding of membrane proteins in the gas phase. Here, we investigate the potential of charge reduction by introducing three membrane protein complexes from saccharide detergents and show how reducing their overall charge enables generation of compact states, as evidenced by ion mobility mass spectrometry. We find that charge reduction stabilizes the oligomeric state and enhances the stability of lipid-bound complexes. This finding is significant since maintaining native-like membrane proteins enables ligand binding to be assessed from a range of detergents that retain solubility while protecting the overall fold.Structural characterization of membrane proteins is challenging due to their hydrophobic nature, low expression levels, and requirement for membrane mimics for solubilization. Amphipathic detergent molecules, which form micelles in solution, are widely adopted for solubilization and their ability to mimic the membrane environment. 1 In a standard nondenaturing mass spectrometry (MS) experiment detergent micelles are removed by collisional activation in the gas phase. 2 Subsequently the folded nature of membrane proteins is assessed using ion-mobility (IM)-MS through measurement of collision cross sections (CCS) and comparison with values calculated from X-ray coordinates. 3 Furthermore, CCS is used to compare candidate models of assemblies to gain structural information about different conformational states. 4,5 It has also been shown that resistance to unfolding, assessed via measuring CCS, can be parametrized and used to rank lipids for their effects on stability. 6 * Corresponding Authorcarol.robinson@chem.ox.ac.uk. Supporting InformationMaterials and methods, Figures S1-S6, and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.. NotesThe authors declare no competing financial interest. For soluble proteins the folded state is affected by combination of the charge state and the voltages applied during acceleration through the mass spectrometer. High charge states are susceptible to Coulombic repulsion between charged residues promoting local unfolding 7 or subunit dissociation. 8 As a consequence of these effects lower charge states are often associated with "native-like" structures. 9 Removal of detergent from membrane protein ions, however, requires activation, and as such, reduction of acceleration voltages is not viable. An alternative strategy is therefore to reduce the charge state of the m...

Intrinsically disordered proteins (IDPs) are important for health and disease, yet their lack of net structure precludes an understanding of their function using classical methods. Gas-phase techniques provide a promising alternative to access information on the structure and dynamics of IDPs, but the fidelity to which these methods reflect the solution conformations of these proteins has been difficult to ascertain. Here we use state of the art ensemble techniques to investigate the solution to gas-phase transfer of a range of different IDPs. We show that IDPs undergo a vast conformational space expansion in the absence of solvent to sample a conformational space 3-5 fold broader than in solution. Moreover, we show that this process is coupled to the electrospray ionization process, which brings about the generation of additional subpopulations for these proteins not observed in solution due to competing effects on protein charge and shape. Ensemble methods have permitted a new definition of the solution to gas-phase transfer of IDPs and provide a roadmap for future investigations into flexible systems by mass spectrometry.

Recent studies have suggested that detergents can protect the structure of membrane proteins during their transition from solution to the gas-phase. Here we provide mechanistic insights into this process by interrogating the structures of membrane protein-detergent assemblies in the gas-phase using ion mobility mass spectrometry. We show a clear correlation between the population of native-like protein conformations and the degree of detergent attachment to the protein in the gas-phase. Interrogation of these protein-detergent assemblies, by tandem mass spectrometry, enables us to define the mechanism by which detergents preserve native-like protein conformations in a solvent free environment. We show that the release of detergent is more central to the survival of these conformations than the physical presence of detergent bound to the protein. We propose that detergent release competes with structural collapse for the internal energy of the ion and permits the observation of transient native-like membrane protein conformations that are otherwise lost to structural rearrangement in the gas-phase.

An investigation into the use of high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to electrospray ionisation mass spectrometry (ESI-MS) for the differentiation of co-populated protein conformers has been conducted on the amyloidogenic protein beta(2)-microglobulin (beta(2)m). Accumulation of beta(2)m in vivo can result in the deposition of insoluble fibrils whose formation is thought to originate from partially folded protein conformers; hence, the folding properties of beta(2)m are of significant interest. We have analysed beta(2)m using ESI-FAIMS-MS under a range of pH conditions and have studied the effect of the ion mobility spectrometry parameters on the behaviour of the various protein conformers. The data show that different protein conformers can be detected and analysed by ESI-FAIMS-MS, the results being consistent with observations of pH denaturation obtained using complementary biophysical techniques. A variant of beta(2)m with different folding characteristics has been analysed for comparison, and the distinctions observed in the data sets for the two proteins are consistent with their folding behaviour. ESI-FAIMS-MS offers significant opportunities for the study of the conformational properties of proteins and thus may present valuable insights into the roles that different conformers play in diseases related to protein folding.

Dialysis-related amyloidosis (DRA) is a complication of hemodialysis where beta2-microglobulin (beta2m) forms plaques mainly in cartilaginous tissues. The tissue-specific deposition, along with a known intransigence of pure beta2m to form fibrils in vitro at neutral pH in the absence of preformed fibrillar seeds, suggests a role for factors within cartilage in enhancing amyloid formation from this protein. To identify these factors, we determined the ability of a derivative lacking the N-terminal six amino acids found in ex vivo beta2m amyloid deposits to form amyloid fibrils at pH 7.4 in the absence of fibrillar seeds. We show that the addition of the glycosaminoglycans (GAGs) chrondroitin-4 or 6-sulfate to fibril growth assays results in the spontaneous generation of amyloid-like fibrils. By contrast, no fibrils are observed over the same time course in the presence of hyaluronic acid, a nonsulfated GAG that is abundant in cartilaginous joints. Based on the observation that hyaluronic acid has no effect on fibril stability, while chrondroitin-6-sulfate decreases the rate of fibril disassembly, we propose that the latter GAG enhances amyloid formation by stabilizing the rare fibrils that form spontaneously. This leads to the accumulation of beta2m in fibrillar deposits. Our data rationalize the joint-specific deposition of beta2m amyloid in DRA, suggesting mechanisms by which amyloid formation may be promoted.

Growing interest in micelles to protect membrane complexes during the transition from solution to gas phase prompts a better understanding of their properties. We have used ion mobility mass spectrometry to separate and assign detergent clusters formed from the n-trimethylammonium bromide series of detergents. We show that cluster size is independent of detergent concentration in solution, increases with charge state, but surprisingly decreases with alkyl chain length. This relationship contradicts the thermodynamics of micelle formation in solution. However, the liquid drop model, which considers both the surface energy and charge, correlates extremely well with the experimental cluster size. To explore further the properties of gas-phase micelles, we have performed collision-induced dissociation on them during tandem mass spectrometry. We observed both sequential asymmetric charge separation and neutral evaporation from the precursor ion cluster. Interestingly, however, we also found markedly different dissociation pathways for the longer alkyl chain detergents, with significantly fewer intermediate ions formed than for those with a shorter alkyl chain. These experiments provide an essential foundation for understanding the process of the gas-phase analysis of membrane protein complexes. Moreover they imply valuable mechanistic details of the protection afforded to protein complexes by detergent clusters during gas-phase activation processes.