The ability of electrospray to propel large viruses into a mass spectrometer is established and rationalized by analogy the atmospheric transmission common cold. Much less clear fate membrane-embedded molecular machines in gas phase. Here we show that rotary adenosine triphosphatases (ATPases)/synthases from Thermus thermophilus Enterococcus hirae can be maintained intact with membrane soluble subunit interactions preserved vacuum. Mass spectra reveal stoichiometries identity tightly bound...

Collapse to compact states in the gas phase, with smaller collision cross sections than calculated for their native-like structure, has been reported previously some protein complexes although not rationalized. Here we combine experimental and theoretical studies investigate gas-phase structures of four multimeric during collisional activation. Importantly, using ion mobility–mass spectrometry (IM–MS), find that all macromolecular retain topologies at low energy. Upon increasing energy, two...

SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including closed state unable to bind the ACE2 receptor, an open that does so but displays more exposed antigenic surface. Spikes variants concern (VOCs) acquired amino acid changes linked increased virulence immune evasion. Here, using HDX-MS, we identified dynamics associate with transition from binding, specific mutations VOCs. We show RBD-associated subdomain...

The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system is an adaptive immune in prokaryotes. Interference complexes encoded by CRISPR-associated (cas) genes utilize small RNAs for homology-directed detection and subsequent degradation of invading genetic elements, they have been classified into three main types (I–III). Type III share the Cas10 subunit but are subclassifed as type IIIA (CSM) IIIB (CMR), depending on their specificity DNA or RNA targets, respectively. role...

Abstract F-type ATPases are highly conserved enzymes used primarily for the synthesis of ATP. Here we apply mass spectrometry to F 1 O -ATPase, isolated from spinach chloroplasts, and uncover multiple modifications in soluble membrane subunits. Mass spectra intact ATPase define a stable lipid ‘plug’ complex reveal stoichiometry nucleotide binding head. Comparing complexes formed solution an untreated with one incubated phosphatase reveals that dephosphorylated enzyme has reduced occupancy...

Current challenges in the field of structural genomics point to need for new tools and technologies obtaining structures macromolecular protein complexes. Here, we present an integrative computational method that uses molecular modelling, ion mobility-mass spectrometry (IM-MS) incomplete atomic structures, usually from X-ray crystallography, generate models subunit architecture We begin by analyzing complexes using IM-MS, taking measurements both intact sub-complexes are generated solution....

Abstract Summary Hydrogen deuterium exchange mass spectrometry (HDX-MS) is becoming increasing routine for monitoring changes in the structural dynamics of proteins. Differential HDX-MS allows comparison protein states, such as absence or presence a ligand. This can be used to attribute conformation binding events, allowing mapping entire conformational networks. As such, number necessary cross-state comparisons quickly increases additional states are introduced system study. There currently...

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the target for neutralizing antibodies elicited following both infection and vaccination. While extensive research has shown that receptor binding domain (RBD) and, to a lesser extent, N-terminal (NTD) are predominant targets antibodies, identification of epitopes beyond these regions important informing vaccine development understanding antibody-mediated immune escape. Here, we identify class broadly bind an epitope on...

Abstract G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation β 1 -adrenergic receptor in response various ligands. By simultaneously capturing effects ligand binding coupling different proteins, probed relative specific...

The role of membrane lipids in modulating eukaryotic transporter assembly and function remains unclear. We investigated the effect structure transport activity purine UapA from Aspergillus nidulans. found that exists mainly as a dimer two lipid molecules bind per dimer. identified three phospholipid classes co-purified with UapA: phosphatidylcholine, phosphatidylethanolamine (PE), phosphatidylinositol (PI). delipidation caused dissociation into monomers. Subsequent addition PI or PE rescued...

Significance Many proteins are located in lipid membranes surrounding cells and subcellular organelles. The membrane can impart important structural functional effects on the protein, making understanding of this interaction critical. Here, we apply computational simulation to identification conserved binding sites an highly bacterial Sec translocase (SecA-SecYEG), which uses ATP proton-motive force (PMF) secrete across plasma membrane. We experimentally validate these use analyses...

Hydrogen deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful technique for interrogating the conformational dynamics of proteins and their complexes. Currently, analysis HDX-MS data remains laborious procedure, mainly due to lack streamlined software process large datasets. We present Deuteros which is standalone designed be coupled with Waters DynamX HDX software, allowing rapid visualization from differential HDX-MS.Deuteros open-source can downloaded...

A yet unresolved challenge in structural biology is to quantify the conformational states of proteins underpinning function. This particularly acute for membrane owing difficulties stabilizing them vitro studies. To address this challenge, we present an integrative strategy that combines hydrogen deuterium exchange–mass spectrometry (HDX-MS) with ensemble modeling. We benchmark our on wild-type and mutant conformers XylE, a prototypical member ubiquitous Major Facilitator Superfamily (MFS)...

Here we examined the gas-phase structures of two tetrameric membrane protein complexes by ion mobility mass spectrometry. The collision cross sections measured for channel are in accord with a compact configuration subunits, suggesting that native-like structure can be preserved under harsh activation conditions required to release it from detergent micelle into gas phase. We also found quaternary transporter, which has fewer transmembrane subunits than channel, is less stable once stripped...

DNA polymerase III, a decameric 420-kDa assembly, simultaneously replicates both strands of the chromosome in Escherichia coli. A subassembly this holoenzyme, seven-subunit clamp loader complex, is responsible for loading sliding (β2) onto DNA. Here, we use structural information derived from ion mobility mass spectrometry (IM-MS) to build three-dimensional models one form full γ3δδ'ψχ (254 kDa). By probing interaction between and single-stranded (ssDNA) binding protein (SSB4) by identifying...

Mass spectrometry (MS) has become an indispensable tool for investigating the architectures and dynamics of macromolecular assemblies. Here we show that covalent labeling solvent accessible residues followed by their MS-based identification yields modeling restraints allow mapping location orientation subunits within protein Together with complementary derived from cross-linking native MS, built native-like models four heterocomplexes known subunit structures compared them available X-ray...

Other than more widely used methods, the use of styrene maleic acid copolymers allows direct extraction membrane proteins from lipid bilayer into SMALPs keeping it in its native surrounding.

Abstract Immunoglobulins are biomolecules involved in defence against foreign substances. Flexibility is key to their functional properties relation antigen binding and receptor interactions. We have developed an integrative strategy combining ion mobility mass spectrometry (IM‐MS) with molecular modelling study the conformational dynamics of human IgG antibodies. Predictive models all four subclasses were assembled sampled transition from extended collapsed state during IM‐MS. Our data...

Transport of proteins across membranes is a fundamental process, achieved in every cell by the ‘Sec’ translocon. In prokaryotes, SecYEG associates with motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed Brownian ratchet model transport, whereby free energy ATP-turnover favours directional diffusion polypeptide (Allen et al., 2016). Here, show that ATP enhances this process modulating secondary structure formation within translocating protein. A...