Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments1-7 and that lipids can bind to specific sites, for example in potassium channels8. Fundamental questions remain however regarding the extent of membrane protein selectivity toward lipids. Here we report a mass spectrometry (MS) approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL), aquaporin Z (AqpZ), and the ammonia channel (AmtB) using ion mobility MS (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 based on their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Results show that lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability, the highest-ranking lipid however is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation9. AqpZ is also stabilized by many lipids with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays, we discover that cardiolipin modulates AqpZ function. Analogous experiments identify AmtB as being highly selective for phosphatidylglycerol prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that reposition AmtB residues to interact with the lipid bilayer. Overall our results demonstrate that resistance to unfolding correlates with specific lipid-binding events enabling distinction of lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be influential not only for defining the selectivity of membrane proteins toward lipids but also for understanding the role of lipids in modulating function or drug binding.

Now routine is the ability to investigate soluble and membrane protein complexes in the gas phase of a mass spectrometer while preserving folded structure and ligand-binding properties. Several recent transformative developments have occurred to arrive at this point. These include advances in mass spectrometry instrumentation, particularly with respect to resolution; the ability to study intact membrane protein complexes released from detergent micelles; and the use of protein unfolding in the gas phase to obtain stability parameters. Together, these discoveries are providing unprecedented information on the compositional heterogeneity of biomacromolecules, the unfolding trajectories of multidomain proteins, and the stability imparted by ligand binding to both soluble and membrane-embedded protein complexes. We review these recent breakthroughs, highlighting the challenges that had to be overcome and the physicochemical insight that can now be gained from studying proteins and their assemblies in the gas phase.

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.

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 invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6–2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.

Multifunctional proteins play a variety of roles in metabolism.Here, we examine the catalytic function of the combined 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM) from Geobacillus sp. DAH7PS operates at the start of the biosynthetic pathway for aromatic metabolites, whereas CM operates in a dedicated branch of the pathway for the biosynthesis of amino acids tyrosine and phenylalanine. In line with sequence predictions, the two catalytic functions are located in distinct domains, and these two activities can be separated and retain functionality. For the full-length protein, prephenate, the product of the CM reaction, acts as an allosteric inhibitor for the DAH7PS. The crystal structure of the full-length protein with prephenate bound and the accompanying small angle x-ray scattering data reveal the molecular mechanism of the allostery. Prephenate binding results in the tighter association between the dimeric CM domains and the tetrameric DAH7PS, occluding the active site and therefore disrupting DAH7PS function. Acquisition of a physical gating mechanism to control catalytic function through gene fusion appears to be a general mechanism for providing allostery for this enzyme.

With the convergence of breakthroughs in structural biology, specifically breaking the resolution barriers in cryo-electron microscopy and with continuing developments in crystallography, novel interfaces with other biophysical methods are emerging. Here we consider how mass spectrometry can inform these techniques by providing unambiguous definition of subunit stoichiometry. Moreover recent developments that increase mass spectral resolution enable molecular details to be ascribed to unassigned density within high-resolution maps of membrane and soluble protein complexes. Importantly we also show how developments in mass spectrometry can define optimal solution conditions to guide downstream structure determination, particularly of challenging biomolecules that refuse to crystallise.

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...