The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either production the amyloid-beta (Abeta) peptides or non-amyloidogenic fragments. Here, we report first structural study C99, 99-residue transmembrane C-terminal domain APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent promotes amyloidogenic pathway, specifically binds this protein. C99 was purified into model membranes where it observed homodimerize....

The feasibility of using solid-state magic-angle-spinning NMR spectroscopy for in situ structural characterization the LR11 (sorLA) transmembrane domain (TM) native Escherichia coli membranes is presented. interacts with human amyloid precursor protein (APP), a central player pathology Alzheimer's disease. background signals from E. lipids and membrane proteins had only minor effects on TM resonances. Approximately 50% residues were assigned by (13)C PARIS data. These assignments allowed...

Significance Despite extensive studies of protein trafficking across length scales many microns, how proteins correctly localize within the smaller bacterial cells is still poorly understood. Recently, we proposed that slight membrane curvature, defined by surface geometry a bacterium, can drive localization certain shape-sensing proteins. Here, developed an assay to quantify curvature recognition small SpoVM and used NMR determine structural basis recognition. molecular dynamics simulations...

A new approach for simultaneous protein backbone resonance assignment and structure determination by NMR is introduced. This relies on recent advances in high-resolution spectroscopy that allow observation of anisotropic interactions, such as dipolar couplings, from proteins partially aligned field ordered media. Residual couplings are used both geometric information a filter the assembly residues sequential manner. Experimental data were collected less than one week small redox protein,...

We present a new approach to the analysis of conformational and motional properties an oligosaccharide, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside. The relies on order matrix residual dipolar couplings in solution state. By combining number different types couplings, (1)D(CH), (2)D(CH), D(HH), is solved for each ring trimannoside. resulting parameters indicate internal motion at alpha (1,3) linkage be limited, while significant suggested (1,6) linkage. Two structures...

Abstract During autophagy the enzyme Atg3 catalyzes covalent conjugation of LC3 to amino group phosphatidylethanolamine (PE) lipids, which is one key steps in autophagosome formation. Here, we have demonstrated that an N-terminal conserved region human (hAtg3) communicates information from membrane curvature-sensitive amphipathic helix (AH), presumably targets tip phagophore, C-terminally located catalytic core for LC3–PE conjugation. Mutations putative communication greatly reduce or...

Autophagosome formation, a crucial step in macroautophagy (autophagy), requires the covalent conjugation of LC3 proteins to amino headgroup phosphatidylethanolamine (PE) lipids. Atg3, an E2-like enzyme, catalyzes transfer from LC3-Atg3 PEs targeted membranes. Here we show that catalytically important C-terminal regions human Atg3 (hAtg3) are conformationally dynamic and directly interact with membrane, collaboration its N-terminal membrane curvature-sensitive helix. The functional relevance...

Abstract VPS37A, an ESCRT-I complex component, is required for recruiting a subset of ESCRT proteins to the phagophore autophagosome closure. However, mechanism by which VPS37A targeted remains obscure. Here, we demonstrate that N-terminal domain exhibits selective interactions with highly curved membranes, mediated two membrane-interacting motifs within disordered regions surrounding its Ubiquitin E2 variant-like (UEVL) domain. Site-directed mutations residues in these disrupt localization...

Vacuolar protein sorting 4 (VPS4) is an AAA-ATPase that catalyzes the endosomal complex required for transport-III disassembly, mediating various cellular membrane-remodeling processes including endolysosomal membrane repair and autophagosome closure. Humans have 2 VPS4 paralogs, VPS4A VPS4B, loss of either paralog has been identified in a significant proportion cancers, rendering them dependent on remaining survival. In this study, we explored inhibition as anticancer strategy by...

Challenges for structural characterization of membrane-bound glycosphingolipids include their high internal dynamic motions and physical proximity to membrane surfaces. Here we demonstrate that NMR paramagnetic relaxation enhancement can be used, alongside independent molecular dynamics simulations an outer-sphere model, quantitatively characterize the presentation (insertion depth orientation relative a surface) ganglioside GM1 in biologically relevant environments. Longitudinal transverse...

Tight junctions (TJs) are dynamic cellular structures that critical for compartmentalizing environments within tissues and regulating transport of small molecules, ions, fluids. Phosphorylation-dependent binding the transmembrane protein occludin to structural organizing ZO-1 contributes regulation barrier properties; however, details their interaction controversial. Using angle X-ray scattering (SAXS), NMR chemical shift perturbation, cross-saturation, in vitro binding, site-directed...

Dormant bacterial spores are encased in a thick protein shell, the 'coat', which contains ∼70 different proteins. The coat protects spore from environmental insults, and is among most durable static structures biology. Owing to extensive cross-linking proteins, this structure has been recalcitrant detailed biochemical analysis, so molecular details of how it assembles largely unknown. Here, we reconstitute basement layer atop spherical membranes supported by silica beads create artificial...

Cations often deform the structure of regulatory proteins to affect a functional response, but for other protein functions more passive effect is desired. For instance, it shown here that in conductance Na+ by gramicidin channel there appears be no significant structural deformation either side chains or backbone upon binding channel. This based on 15N and 13C chemical shifts, 2H quadrupolar interactions, 15N−2H dipolar interactions obtained solid-state NMR spectroscopy uniformly aligned...

Meprin metalloproteases are highly expressed at the luminal interface of intestine and kidney in certain leukocytes. Meprins cleave a variety substrates vitro, including extracellular matrix proteins, adherens junction cytokines, have been implicated number inflammatory diseases. The linkage between results vitro pathogenesis, however, has not elucidated. present study aimed to determine whether meprins determinative factors disrupting barrier function epithelium. Active meprin A or B...

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTIntensity-Based Measurement of Homonuclear Residual Dipolar Couplings from CT-COSYFang Tian, Pascal J. Bolon, and H. PrestegardView Author Information Complex Carbohydrate Research Center University Georgia, Athens, Georgia 30602 Cite this: Am. Chem. Soc. 1999, 121, 33, 7712–7713Publication Date (Web):August 10, 1999Publication History Received2 April 1999Revised29 June 1999Published online10 August inissue 1...

An exchange is better than a rest: NMR screening method described which facilitates the of compound mixtures for components that bind protein drug targets. The approach allows detection active molecules thanks to "probe" molecule, competes with library potential ligands binding sites on targeted protein. whole spectrum ligands, including those displaying either very fast or slow behavior, are equally susceptible by this method.

Functional regulation of proteins is central to living organisms. Here it shown that a nonfunctional conformational state polypeptide can be kinetically trapped in lipid bilayer environment. This metastable structure stable for weeks just above the phase transition temperature lipid. When samples are incubated several days at 68 degrees C, 50% conformation converts minimum-energy functional state. result suggests possibility another mechanism protein activity may available membrane proteins:...

We present a nuclear magnetic resonance (NMR) study in solution of the structures human normal hemoglobin (Hb A) deoxy or unligated form absence and presence an allosteric effector, inositol hexaphosphate (IHP), using 15N−1H residual dipolar coupling (RDC) measurements. There are several published crystal for deoxyhemoglobin A (deoxy-Hb A), it has been reported that functional properties Hb single crystals different from those solution. Carbonmonoxyhemoglobin (HbCO can also be crystallized...