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      Probing the solution structure of the E. coli multidrug transporter MdfA using DEER distance measurements with nitroxide and Gd(III) spin labels

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          Abstract

          Methodological and technological advances in EPR spectroscopy have enabled novel insight into the structural and dynamic aspects of integral membrane proteins. In addition to an extensive toolkit of EPR methods, multiple spin labels have been developed and utilized, among them Gd(III)-chelates which offer high sensitivity at high magnetic fields. Here, we applied a dual labeling approach, employing nitroxide and Gd(III) spin labels, in conjunction with Q-band and W-band double electron-electron resonance (DEER) measurements to characterize the solution structure of the detergent-solubilized multidrug transporter MdfA from E. coli. Our results identify highly flexible regions of MdfA, which may play an important role in its functional dynamics. Comparison of distance distribution of spin label pairs on the periplasm with those calculated using inward- and outward-facing crystal structures of MdfA, show that in detergent micelles, the protein adopts a predominantly outward-facing conformation, although more closed than the crystal structure. The cytoplasmic pairs suggest a small preference to the outward-facing crystal structure, with a somewhat more open conformation than the crystal structure. Parallel DEER measurements with the two types of labels led to similar distance distributions, demonstrating the feasibility of using W-band spectroscopy with a Gd(III) label for investigation of the structural dynamics of membrane proteins.

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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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            Membrane-bound alpha-synuclein forms an extended helix: long-distance pulsed ESR measurements using vesicles, bicelles, and rodlike micelles.

            We apply pulsed dipolar ESR spectroscopy (Ku-band DEER) to elucidate the global conformation of the Parkinson's disease-associated protein, alpha-synuclein (alphaS) bound to small unilamellar phospholipid vesicles, rodlike SDS micelles, or lipid bicelles. By measuring distances as long as approximately 7 nm between introduced pairs of nitroxide spin labels, we show that distances are close to the expectations for a single continuous helix in all cases studied. In particular, we find distances of 7.5 nm between sites 24 and 72; 5.5 nm between sites 24 and 61; and 2 nm between sites 35 and 50. We conclude that alphaS does not retain a "hairpin" structure with two antiparallel helices, as is known to occur with spheroidal micelles, in agreement with our earlier finding that the protein's geometry is determined by the surface topology rather than being constrained by the interhelix linker. While the possibility of local helix discontinuities in the structure of membrane-bound alphaS remains, our data are more consistent with one intact helix. Importantly, we demonstrate that bicelles produce very similar results to liposomes, while offering a major improvement in experimentally accessible distance range and resolution, and thus are an excellent lipid membrane mimetic for the purpose of pulse dipolar ESR spectroscopy.
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              Probing Protein Conformation in Cells by EPR Distance Measurements using Gd3+ Spin Labeling

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                Author and article information

                Contributors
                eitan.bibi@weizmann.ac.il
                hassane.mchaourab@vanderbilt.edu
                Daniella.goldfarb@weizmann.ac.il
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                29 August 2019
                29 August 2019
                2019
                : 9
                : 12528
                Affiliations
                [1 ]ISNI 0000 0004 0604 7563, GRID grid.13992.30, Department of Biomolecular Sciences, , Weizmann Institute of Science Rehovot, ; Rehovot, 76100 Israel
                [2 ]ISNI 0000 0004 0604 7563, GRID grid.13992.30, Department of Chemical and Biological Physics, , Weizmann Institute of Science, ; Rehovot, 76100 Israel
                [3 ]ISNI 0000 0001 2264 7217, GRID grid.152326.1, Department of Molecular Physiology and Biophysics, , Vanderbilt University, ; Nashville, TN USA
                [4 ]ISNI 0000 0004 1936 7857, GRID grid.1002.3, Monash Institute of Pharmaceutical Sciences, , Monash University, ; Parkville, VIC 3052 Australia
                [5 ]ISNI 0000 0004 1936 7857, GRID grid.1002.3, School of Chemistry, , Monash University, ; Wellington Road, Clayton, Victoria Australia
                [6 ]ISNI 0000 0001 2180 7477, GRID grid.1001.0, Research School of Chemistry, , The Australian National University, ; Canberra, ACT 2601 Australia
                Author information
                http://orcid.org/0000-0001-5714-7159
                Article
                48694
                10.1038/s41598-019-48694-0
                6715713
                31467343
                14b28948-d472-49c5-aa08-dbc78bdb7502
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 22 April 2019
                : 8 August 2019
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100001658, Minerva Foundation (Minerva Stiftung);
                Award ID: not applicable
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/501100001742, United States-Israel Binational Science Foundation (BSF);
                Award ID: 2017081
                Award Recipient :
                Categories
                Article
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                © The Author(s) 2019

                Uncategorized
                biophysical chemistry,biophysics
                Uncategorized
                biophysical chemistry, biophysics

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