HDX-guided EPR spectroscopy to interrogate membrane protein dynamics

This project was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/S018069/1) to C.P., who also acknowledges support from the Wellcome Trust (WT) (219999/Z/19/Z) and the Chinese Scholarship Council (CSC) in the form of studentships for B.J.L. and B.W., respectively. A.N.C. is a Sir Henry Dale Fellow jointly funded by the WT and the Royal Society (220628/Z/20/Z). Funding from the BBSRC (BB/M012573/1) enabled the purchase of mass spectrometry equipment.Solvent accessibilities of and distances between protein residues measured by pulsed-EPR approaches provide high-resolution information on dynamic protein motions. We describe protocols for the purification and site-directed spin labeling of integral membrane proteins. In our protocol, peptide-level HDX-MS is used as a precursor to guide single-residue resolution ESEEM accessibility measurements and spin labeling strategies for EPR applications. Exploiting the pentameric MscL channel as a model, we discuss the use of cwEPR, DEER/PELDOR, and ESEEM spectroscopies to interrogate membrane protein dynamics. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).Publisher PDFPeer reviewe

Investigating the structural compaction of biomolecules upon transition to the gas-phase using ESI-TWIMS-MS

Collision cross-section (CCS) measurements obtained from ion mobility spectrometry-mass spectrometry (IMS-MS) analyses often provide useful information concerning a protein’s size and shape and can be complemented by modeling procedures. However, there have been some concerns about the extent to which certain proteins maintain a native-like conformation during the gas-phase analysis, especially proteins with dynamic or extended regions. Here we have measured the CCSs of a range of biomolecules including non-globular proteins and RNAs of different sequence, size, and stability. Using traveling wave IMS-MS, we show that for the proteins studied, the measured CCS deviates significantly from predicted CCS values based upon currently available structures. The results presented indicate that these proteins collapse to different extents varying on their elongated structures upon transition into the gas-phase. Comparing two RNAs of similar mass but different solution structures, we show that these biomolecules may also be susceptible to gas-phase compaction. Together, the results suggest that caution is needed when predicting structural models based on CCS data for RNAs as well as proteins with non-globular folds

Structural basis of rotavirus RNA chaperone displacement and RNA annealing.

Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling

Structural mapping of oligomeric intermediates in an amyloid assembly pathway

Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein ΔN6, a truncation variant of human β2-microglobulin (β2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-β structure formation. The results provide a detailed structural view of early amyloid assembly involving structured species that are not cytotoxic

Skp is a multivalent chaperone of outer membrane proteins

The trimeric chaperone Skp sequesters outer-membrane proteins (OMPs) within a hydrophobic cage, thereby preventing their aggregation during transport across the periplasm in Gram-negative bacteria. Here, we studied the interaction between Escherichia coli Skp and five OMPs of varying size. Investigations of the kinetics of OMP folding revealed that higher Skp/OMP ratios are required to prevent the folding of 16-stranded OMPs compared with their 8-stranded counterparts. Ion mobility spectrometry–mass spectrometry (IMS–MS) data, computer modeling and molecular dynamics simulations provided evidence that 10- to 16-stranded OMPs are encapsulated within an expanded Skp substrate cage. For OMPs that cannot be fully accommodated in the expanded cavity, sequestration is achieved by binding of an additional Skp trimer. The results suggest a new mechanism for Skp chaperone activity involving the coordination of multiple copies of Skp in protecting a single substrate from aggregation

From Geometry to Numerics: interdisciplinary aspects in mathematical and numerical relativity

This article reviews some aspects in the current relationship between mathematical and numerical General Relativity. Focus is placed on the description of isolated systems, with a particular emphasis on recent developments in the study of black holes. Ideas concerning asymptotic flatness, the initial value problem, the constraint equations, evolution formalisms, geometric inequalities and quasi-local black hole horizons are discussed on the light of the interaction between numerical and mathematical relativists.Comment: Topical review commissioned by Classical and Quantum Gravity. Discussion inspired by the workshop "From Geometry to Numerics" (Paris, 20-24 November, 2006), part of the "General Relativity Trimester" at the Institut Henri Poincare (Fall 2006). Comments and references added. Typos corrected. Submitted to Classical and Quantum Gravit

Bimodal action of the flavonoid quercetin on basophil function: an investigation of the putative biochemical targets

<p>Abstract</p> <p>Background</p> <p>Flavonoids, a large group of polyphenolic metabolites derived from plants have received a great deal of attention over the last several decades for their properties in inflammation and allergy. Quercetin, the most abundant of plant flavonoids, exerts a modulatory action at nanomolar concentrations on human basophils. As this mechanism needs to be elucidated, in this study we focused the possible signal transduction pathways which may be affected by this compound. Methods: K2-EDTA derived leukocyte buffy coats enriched in basophil granulocytes were treated with different concentrations of quercetin and triggered with anti-IgE, fMLP, the calcium ionophore A23187 and the phorbol ester PMA in different experimental conditions. Basophils were captured in a flow cytometry analysis as CD123bright/HLADRnon expressing cells and fluorescence values of the activation markers CD63-FITC or CD203c-PE were used to produce dose response curves. The same population was assayed for histamine release.</p> <p>Results</p> <p>Quercetin inhibited the expression of CD63 and CD203c and the histamine release in basophils activated with anti-IgE or with the ionophore: the IC50 in the anti-IgE model was higher than in the ionophore model and the effects were more pronounced for CD63 than for CD203c. Nanomolar concentrations of quercetin were able to prime both markers expression and histamine release in the fMLP activation model while no effect of quercetin was observed when basophils were activated with PMA. The specific phosphoinositide-3 kinase (PI3K) inhibitor wortmannin exhibited the same behavior of quercetin in anti-IgE and fMLP activation, thus suggesting a role for PI3K involvement in the priming mechanism.</p> <p>Conclusions</p> <p>These results rule out a possible role of protein kinase C in the complex response of basophil to quercetin, while indirectly suggest PI3K as the major intracellular target of this compound also in human basophils.</p

Systematic analysis of the use of amphipathic polymers for studies of outer membrane proteins using mass spectrometry

Membrane proteins (MPs) are essential for numerous important biological processes. Recently, mass spectrometry (MS), coupled with an array of related techniques, has been used to probe the structural properties of MPs and their complexes. Typically, detergent micelles have been employed for delivering MPs into the gas-phase, but these complexes have intrinsic properties that can limit the utility of structural studies of MPs using MS methods. Amphipols (APols) have advantages over detergent micelles and have been shown to be capable of delivering native MPs into the gas-phase. Comparing six different APols which vary in mass and charge, and the detergent n-dodecyl-β-d-maltopyranoside, we aimed to determine which APols are most efficient for delivery of native outer membrane proteins (OMPs) into the gas-phase. We show that maintaining the solution-phase folding and global structures of three different OMPs (PagP, OmpT and tOmpA) are independent of the APol used, but differences in OMP activity can result from the different APol:OMP complexes. ESI-IMS-MS analysis of OMP:APol complexes shows that the A8-35 APol is most proficient at liberating all three OMPs into the gas-phase, without altering their gas-phase conformations