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      Electron Paramagnetic Resonance as a Tool for Studying Membrane Proteins

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          Abstract

          Membrane proteins possess a variety of functions essential to the survival of organisms. However, due to their inherent hydrophobic nature, it is extremely difficult to probe the structure and dynamic properties of membrane proteins using traditional biophysical techniques, particularly in their native environments. Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a very powerful and rapidly growing biophysical technique to study pertinent structural and dynamic properties of membrane proteins with no size restrictions. In this review, we will briefly discuss the most commonly used EPR techniques and their recent applications for answering structure and conformational dynamics related questions of important membrane protein systems.

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          Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms.

          We have carried out detailed statistical analyses of integral membrane proteins of the helix-bundle class from eubacterial, archaean, and eukaryotic organisms for which genome-wide sequence data are available. Twenty to 30% of all ORFs are predicted to encode membrane proteins, with the larger genomes containing a higher fraction than the smaller ones. Although there is a general tendency that proteins with a smaller number of transmembrane segments are more prevalent than those with many, uni-cellular organisms appear to prefer proteins with 6 and 12 transmembrane segments, whereas Caenorhabditis elegans and Homo sapiens have a slight preference for proteins with seven transmembrane segments. In all organisms, there is a tendency that membrane proteins either have many transmembrane segments with short connecting loops or few transmembrane segments with large extra-membraneous domains. Membrane proteins from all organisms studied, except possibly the archaeon Methanococcus jannaschii, follow the so-called "positive-inside" rule; i.e., they tend to have a higher frequency of positively charged residues in cytoplasmic than in extra-cytoplasmic segments.
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            Dead-time free measurement of dipole-dipole interactions between electron spins.

            A four-pulse version of the pulse double electron-electron resonance (DEER) experiment is presented, which is designed for the determination of interradical distances on a nanoscopic length-scale. With the new pulse sequence electron-electron couplings can be studied without dead-time artifacts, so that even broad distributions of electron-electron distances can be characterized. A version of the experiment that uses a pulse train in the detection period exhibits improved signal-to-noise ratio. Tests on two nitroxide biradicals with known length indicate that the accessible range of distances extends from about 1.5 to 8 nm. The four-pulse DEER spectra of an ionic spin probe in an ionomer exhibit features due to probe molecules situated both on the same and on different ion clusters. The former feature provides information on the cluster size and is inaccessible with previous methods. Copyright 2000 Academic Press.
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              The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol.

              C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer's disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded "N-helix" followed by a short "N-loop" connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer's therapeutics.
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                Author and article information

                Journal
                Biomolecules
                Biomolecules
                biomolecules
                Biomolecules
                MDPI
                2218-273X
                13 May 2020
                May 2020
                : 10
                : 5
                : 763
                Affiliations
                [1 ]Natural Science Division, Campbellsville University, Campbellsville, KY 42718, USA
                [2 ]Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA
                Author notes
                [* ]Correspondence: idsahu@ 123456campbellsville.edu (I.D.S.); lorigag@ 123456miamioh.edu (G.A.L.); Tel.: +(270)-789-5597 (I.D.S.); Tel.: +(513)-529-3338 (G.A.L.)
                Article
                biomolecules-10-00763
                10.3390/biom10050763
                7278021
                32414134
                1212cc8a-8bf0-4f2d-a7c7-cd18befbc875
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 29 January 2020
                : 24 April 2020
                Categories
                Review

                membrane protein,electron paramagnetic resonance (epr),site-directed spin labeling,structural and dynamics,membrane mimetic,double electron electron resonance (deer)

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