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      Deuteration Aiming for Neutron Scattering


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          The distinguished feature of neutron as a scattering probe is an isotope effect, especially the large difference in neutron scattering length between hydrogen and deuterium. The difference renders the different visibility between hydrogenated and deuterated proteins. Therefore, the combination of deuterated protein and neutron scattering enables the selective visualization of a target domain in the complex or a target protein in the multi-component system. Despite of this fascinating character, there exist several problems for the general use of this method: difficulty and high cost for protein deuteration, and control and determination of deuteration ratio of the sample. To resolve them, the protocol of protein deuteration techniques is presented in this report. It is strongly expected that this protocol will offer more opportunity for conducting the neutron scattering studies with deuterated proteins.

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          Most cited references 18

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          Mechanical Properties of Nanoscopic Lipid Domains

          The lipid raft hypothesis presents insights into how the cell membrane organizes proteins and lipids to accomplish its many vital functions. Yet basic questions remain about the physical mechanisms that lead to the formation, stability, and size of lipid rafts. As a result, much interest has been generated in the study of systems that contain similar lateral heterogeneities, or domains. In the current work we present an experimental approach that is capable of isolating the bending moduli of lipid domains. This is accomplished using neutron scattering and its unique sensitivity to the isotopes of hydrogen. Combining contrast matching approaches with inelastic neutron scattering, we isolate the bending modulus of ∼13 nm diameter domains residing in 60 nm unilamellar vesicles, whose lipid composition mimics the mammalian plasma membrane outer leaflet. Importantly, the bending modulus of the nanoscopic domains differs from the modulus of the continuous phase surrounding them. From additional structural measurements and all-atom simulations, we also determine that nanoscopic domains are in-register across the bilayer leaflets. Taken together, these results inform a number of theoretical models of domain/raft formation and highlight the fact that mismatches in bending modulus must be accounted for when explaining the emergence of lateral heterogeneities in lipid systems and biological membranes.
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            Efficient segmental isotope labeling of multi-domain proteins using Sortase A.

            NMR studies of multi-domain protein complexes provide unique insight into their molecular interactions and dynamics in solution. For large proteins domain-selective isotope labeling is desired to reduce signal overlap, but available methods require extensive optimization and often give poor ligation yields. We present an optimized strategy for segmental labeling of multi-domain proteins using the S. aureus transpeptidase Sortase A. Critical improvements compared to existing protocols are (1) the efficient removal of cleaved peptide fragments by centrifugal filtration and (2) a strategic design of cleavable and non-cleavable affinity tags for purification. Our approach enables routine production of milligram amounts of purified segmentally labeled protein for NMR and other biophysical studies.
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              Effects of macromolecular crowding on an intrinsically disordered protein characterized by small-angle neutron scattering with contrast matching.

              Small-angle neutron scattering was used to examine the effects of molecular crowding on an intrinsically disordered protein, the N protein of bacteriophage λ, in the presence of high concentrations of a small globular protein, bovine pancreatic trypsin inhibitor (BPTI). The N protein was labeled with deuterium, and the D(2)O concentration of the solvent was adjusted to eliminate the scattering contrast between the solvent and unlabeled BPTI, leaving only the scattering signal from the unfolded protein. The scattering profile observed in the absence of BPTI closely matched that predicted for an ensemble of random conformations. With BPTI added to a concentration of 65 mg/mL, there was a clear change in the scattering profile representing an increase in the mass fractal dimension of the unfolded protein, from 1.7 to 1.9, as expected if crowding favors more compact conformations. The crowding protein also inhibited aggregation of the unfolded protein. At 130 mg/mL BPTI, however, the fractal dimension was not significantly different from that measured at the lower concentration, contrary to the predictions of models that treat the unfolded conformations as convex particles. These results are reminiscent of the behavior of polymers in concentrated melts, suggesting that these synthetic mixtures may provide useful insights into the properties of unfolded proteins under crowding conditions. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

                Author and article information

                Biophys Physicobiol
                Biophys Physicobiol
                Biophysics and Physicobiology
                The Biophysical Society of Japan
                6 February 2021
                : 18
                : 16-27
                [1 ] Institute for Integrated Radiation and Nuclear Science, Kyoto University , Sennan-gun, Osaka 590-0494 Japan
                [2 ] Institute of Advanced Medical Sciences, Tokushima University , Tokushima 770-8503, Japan
                [3 ] Graduate School of Pharmaceutical Sciences, Nagoya City University , Nagoya, Aichi 467-8603, Japan
                [4 ] Institute for Molecular Science (IMS), National Institutes of Natural Sciences , Okazaki, Aichi 444-8787, Japan
                [5 ] Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences , Okazaki, Aichi 444-8787, Japan
                Author notes

                Edited by Koichiro Ishimori


                These authors contributed equally to this work.

                Corresponding authors: Rintaro Inoue, Institute for Integrated Radiation and Nuclear Science, Kyoto University, 2-1010 Asashiro Nishi, Kumatori, Sennan-gun, Osaka 590-0494 Japan. e-mail: inoue.rintaro.5w@ 123456kyoto-u.ac.jp ; Masaaki Sugiyama, Institute for Integrated Radiation and Nuclear Science, Kyoto University, 2-1010 Asashiro Nishi, Kumatori, Sennan-gun, Osaka 590-0494 Japan. e-mail: sugiyama.masaaki.5n@ 123456kyoto-u.ac.jp
                JST.JSTAGE/biophysico/bppb-v18.003 bppb-v18.003

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