Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy |
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Authors: | Donghua H Zhou Andrew J Nieuwkoop Deborah A Berthold Gemma Comellas Lindsay J Sperling Ming Tang Gautam J Shah Elliott J Brea Luisel R Lemkau Chad M Rienstra |
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Institution: | 1. Department of Physics, Oklahoma State University, Stillwater, OK, 74074, USA 2. Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA 3. Leibniz-Institut f??r Molekulare Pharmakologie, 13125, Berlin, Germany 4. Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA 5. Materials Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA
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Abstract: | Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6?kDa), a deuterated microcrystalline protein (DsbA, 21?kDa), a membrane protein (DsbB, 20?kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (??-synuclein, 14?kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100?% amide proton), fast magic-angle spinning conditions (40?kHz) and moderate proton decoupling power levels. Each H?CN pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution. |
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