Title | Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy. |
Publication Type | Journal Article |
Year of Publication | 2012 |
Authors | Zhou DH, Nieuwkoop AJ, Berthold DA, Comellas G, Sperling LJ, Tang M, Shah GJ, Brea EJ, Lemkau LR, Rienstra CM |
Journal | J Biomol NMR |
Volume | 54 |
Issue | 3 |
Pagination | 291-305 |
Date Published | 2012 Nov |
ISSN | 1573-5001 |
Keywords | alpha-Synuclein, Bacterial Proteins, Deuterium, Escherichia coli Proteins, Membrane Proteins, Nuclear Magnetic Resonance, Biomolecular, Protein Disulfide-Isomerases, Protons |
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 (α-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-N 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. |
DOI | 10.1007/s10858-012-9672-z |
Alternate Journal | J. Biomol. NMR |
PubMed ID | 22986689 |
PubMed Central ID | PMC3484199 |
Grant List | R15 GM097713 / GM / NIGMS NIH HHS / United States R01 GM073770 / GM / NIGMS NIH HHS / United States R01 GM075937 / GM / NIGMS NIH HHS / United States R01 GM-75937 / GM / NIGMS NIH HHS / United States R15 GM-097713 / GM / NIGMS NIH HHS / United States R01 GM-73770 / GM / NIGMS NIH HHS / United States |
Submitted by kej2006 on June 6, 2018 - 4:09pm