1,720,991 research outputs found
Structural mass spectrometry of membrane proteins within their native lipid environments
No full textMass 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 highlighte
Structural mass spectrometry approaches to understand multidrug efflux systems
Get PDFMultidrug 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
Using hydrogen/deuterium exchange mass spectrometry to understand bacterial membrane efflux proteins
No full textBacterial multidrug efflux pumps play major roles in antibiotic and multidrug resistance as well as fulfilling many important physiological functions. These molecular machines are highly dynamic, with structural malleability at the core of their action, and elaborating their conformational features can provide insight into the mechanisms underpinning their activities and responses to transport substrates and inhibitors. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a structural biology assay used to monitor protein backbone dynamics and is capable of elucidating conformational signatures that define function and fold. In recent years, HDX-MS methods have advanced, making its use more amenable to the study of membrane proteins, yet experimental challenges remain. In this chapter, we provide background on HDX-MS, its limitations, and discussions around experimental design and optimization for studying efflux pump proteins, including its application to study membrane proteins in lipid environments. To demonstrate its utility, we provide an original case study on AcrB inhibition by an efflux pump inhibitor (MBX-3756), employing membrane-scaffold protein lipid nanodiscs, revealing perturbation of backbone motions distinguishing of hydrophobic-trap targeting inhibitory action. This chapter can serve as a guide for designing HDX-MS investigations on efflux pump proteins and membrane proteins in general.</p
Quantifying the stabilizing effects of protein-ligand interactions in the gas phase.
Get PDFThe 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
Peptide-based approach to inhibition of the multidrug resistance efflux pump AcrB
Get PDFClinically 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 Förster 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
Cell-Free Synthesis Strategies to Probe Co-translational Folding of Proteins Within Lipid Membranes
No full textIn 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
Silver and Palladium Complexes Containing Ditopic N-Heterocyclic Carbene-Thione Ligands
No full textThe 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)(L-2)][X](2) and [PdX(CSMe)(L-2)][X] (where L-2 = 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.</p
Native mass spectrometry goes more native: investigation of membrane protein complexes directly from SMALPs
Get PDFOther 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.</p
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