Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy.

TitleSolid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy.
Publication TypeJournal Article
Year of Publication2012
AuthorsZhou DH, Nieuwkoop AJ, Berthold DA, Comellas G, Sperling LJ, Tang M, Shah GJ, Brea EJ, Lemkau LR, Rienstra CM
JournalJ Biomol NMR
Volume54
Issue3
Pagination291-305
Date Published2012 Nov
ISSN1573-5001
Keywordsalpha-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.

DOI10.1007/s10858-012-9672-z
Alternate JournalJ. Biomol. NMR
PubMed ID22986689
PubMed Central IDPMC3484199
Grant ListR15 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

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