Biology subjects

Gault, J.

Publications and source records attributed to Gault, J..

7 recordsLinked to original sources

bioRxiv2023

Cryo-EM of soft-landed β-galactosidase: Gas-phase and native structures are remarkably similar

Native mass spectrometry (native MS) is a powerful technique that provides information on stoichiometry, interactions, homogeneity and shape of protein complexes. However, the extent of deviation between protein structures in the mass spectrometer and in solution remains a matter of debate. Here, we uncover the gas-phase structure of {beta}-galactosidase using single particle electron cryomicroscopy (cryo-EM) down to 2.6 [A] resolution, enabled by soft-landing of mass-selected protein complexes onto cold TEM grids and in-situ ice coating. We find that large parts of the secondary and tertiary structure are retained from solution, with dehydration-driven subunit reorientation leading to consistent compaction in the gas phase. Our work enables visualizing the structure of gas-phase protein com-plexes from numerous experimental scenarios at side-chain resolution and demonstrates the possibility of more controlled cryo-EM sample preparation. One Sentence SummaryElectrospray ion-beam deposition on cold grids and in-vacuum ice growth enable cryo-EM of mass-selected proteins at 2.6 [A].

biochemistry↗

bioRxiv2022

Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system

Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for both this phage and a cos system, reveal unexpected mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.

biochemistry↗

bioRxiv2021

Mass-selective and ice-free cryo-EM protein sample preparation via native electrospray ion-beam deposition

Electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA) have revolutionized structure determination of homogeneous proteins. However, obtaining high-resolution structures from heterogeneous samples remains a major challenge, as the various protein states embedded in thin films of vitreous ice may be classified incorrectly, resulting in detrimental averaging of features. Here we present native electrospray ion-beam deposition (native ES-IBD) for the preparation of extremely high-purity cryo-EM samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, mass-filtered, and gently deposited on cryo-EM grids, and subsequently frozen in liquid nitrogen. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected proteins and protein assemblies. SPA reveals that they remain structurally intact, but variations in secondary and tertiary structure are currently limiting information in 2D classes and 3D EM density maps. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM structure determination.

molecular biology↗

bioRxiv2020

Correlating glycoforms of DC-SIGN with stability using a combination of enzymatic digestion and ion mobility MS

The immune scavenger protein DC-SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC-SIGN have not been reported. Here, we develop and apply a mass spectrometry-based approach to both uncover and characterise the effects of O-glycans on the stability of DC-SIGN. We first quantify the Core 1 & 2 O-glycan structures on the carbohydrate recognition and extracellular domains of the protein via sequential exoglycosidase sequencing. We then use ion mobility mass spectrometry to show how specific O-glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas-phase stability of the glycoprotein isoforms of DC-SIGN. We find that rather than the mass or length of glycoprotein modifications, the stability of DC-SIGN is better correlated with the number of glycosylation sites. Collectively, our results exemplify a combined multi-dimensional MS approach, proficient in evaluating protein stability in response to both glycoprotein macro- and micro-heterogeneity and adding structural detail to the infection enhancer DC-SIGN.

biophysics↗

bioRxiv2020

Mass Photometry of Membrane Proteins

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane mimetic systems at the single particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterising the particle size, sample purity and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers - in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.

biophysics↗

bioRxiv2019

Pyocin S5 import into Pseudomonas aeruginosa reveals a generic mode of bacteriocin transport

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen P. aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analysis and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked three helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli towards PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria.

biochemistry↗

bioRxiv2018

Structure of the Core of the Type Three Secretion System ExportApparatus

Export of proteins through type three secretion systems is critical for bacterial motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP/FliQ/FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. We here present the structure of this complex at 4.2 [A] by cryo-electron microscopy. None of the subunits adopt canonical integral membrane protein topologies and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems localize the export gate as a core component of the periplasmic portion of the machinery, and cross-linking experiments confirm this observation. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.\n\nOne Sentence SummaryThe core of the T3SS export gate forms a supra-membrane helical assembly

microbiology↗