Fast-time scale dynamics of outer membrane protein A by extended model-free analysis of NMR relaxation data

AbstractIn order to better understand the dynamics of an integral membrane protein, backbone amide 15N NMR dynamics measurements of the β-barrel membrane protein OmpA have been performed at three magnetic fields. A total of nine relaxation data sets were globally analyzed using an extended model-free formalism. The diffusion tensor was found to be prolate axially symmetric with an axial ratio of 5.75, indicating a possible rotation of the protein within the micelle. The generalized order parameters gradually decreased from the mid-plane towards the two ends of the barrel, counteracting the dynamic gradient of the lipids in a matching bilayer, and were dramatically reduced in the extracellular loops. Large-scale internal motions on the ns time scale indicate that entire loops most likely undergo concerted (“sea anemone”-like) motions emanating from their anchoring points on the barrel. The case of OmpA in DPC micelles also illustrates inherent limitations of analyzing the data with even the most sophisticated current models of the model-free formalism. It is likely that conformational exchange processes on the ms–μs also play a role in describing the motions of some residues, but their analysis did not produce unique results that could be independently verified

Role of Sequence and Structure of the Hendra Fusion Protein Fusion Peptide in Membrane Fusion

Viral fusion proteins are intriguing molecular machines that undergo drastic conformational changes to facilitate virus-cell membrane fusion. During fusion a hydrophobic region of the protein, termed the fusion peptide (FP), is inserted into the target host cell membrane, with subsequent conformational changes culminating in membrane merger. Class I fusion proteins contain FPs between 20 and 30 amino acids in length that are highly conserved within viral families but not between. To examine the sequence dependence of the Hendra virus (HeV) fusion (F) protein FP, the first eight amino acids were mutated first as double, then single, alanine mutants. Mutation of highly conserved glycine residues resulted in inefficient F protein expression and processing, whereas substitution of valine residues resulted in hypofusogenic F proteins despite wild-type surface expression levels. Synthetic peptides corresponding to a portion of the HeV F FP were shown to adopt an α-helical secondary structure in dodecylphosphocholine micelles and small unilamellar vesicles using circular dichroism spectroscopy. Interestingly, peptides containing point mutations that promote lower levels of cell-cell fusion within the context of the whole F protein were less α-helical and induced less membrane disorder in model membranes. These data represent the first extensive structure-function relationship of any paramyxovirus FP and demonstrate that the HeV F FP and potentially other paramyxovirus FPs likely require an α-helical structure for efficient membrane disordering and fusion

Electrophysiology of Concatameric Pannexin 1 Channels Reveals the Stoichiometry of C-Terminal Autoinhibition

Codi d'Art Públic: 8008-1 (La República); Reportatge realitzat als dies 4 i 18-7-1990Pericas, Enric (arquitecte); Viaplana, Albert (arquitecte i estructura); Viladomat Massanas, Josep (escultura);Joan Pie (Medalló); Piñón, Helio (Estr

Synaptotagmin‐7 enhances calcium‐sensing of chromaffin cell granules and slows discharge of granule cargos

Synaptotagmin‐7 (Syt‐7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin‐1 (Syt‐1). Despite a broad appreciation for the importance of Syt‐7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations—mouse chromaffin cells lacking endogenous Syt‐7 (KO cells) and a reconstituted system employing cell‐derived granules expressing either Syt‐7 or Syt‐1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt‐7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt‐7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt‐1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt‐7, granules expressing only Syt‐7 or Syt‐1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt‐7 confers substantially greater calcium sensitivity to granule fusion than Syt‐1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt‐7 plays a central role in regulating secretory output from adrenal chromaffin cells.Syt‐7 is a high‐affinity calcium sensor expressed on chromaffin cell dense core granules. The purpose of this study was to assess the role of Syt‐7 in regulating the secretory response to cholinergic stimulation. Acetylcholine elicits secretion by elevating cytosolic calcium. The calcium sensitivity of exocytosis in cells lacking Syt‐7 is impaired. Cells that lack Syt‐7 also release peptide hormones at faster rates, implicating a role for Syt‐7 in regulating the exocytotic fusion pore. These data demonstrate that Syt‐7 has an important role in triggering exocytosis in cells and is likely to play a role in controlling hormone output, in situ.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162737/3/jnc14986.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162737/2/jnc14986-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162737/1/jnc14986_am.pd

Protein-Lipid Interactions From Membrane Domains To Cellular Networks

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