title: Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy.
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.
authors.affiliation: Department of Physics, Oklahoma State University, Stillwater, OK 74074, USA. donghua@okstate.edu
authors.forename: Donghua H
authors.initials: DH
authors.lastname: Zhou
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Nieuwkoop
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Berthold
authors.forename: Gemma
authors.initials: G
authors.lastname: Comellas
authors.forename: Lindsay J
authors.initials: LJ
authors.lastname: Sperling
authors.forename: Ming
authors.initials: M
authors.lastname: Tang
authors.forename: Gautam J
authors.initials: GJ
authors.lastname: Shah
authors.forename: Elliott J
authors.initials: EJ
authors.lastname: Brea
authors.forename: Luisel R
authors.initials: LR
authors.lastname: Lemkau
authors.forename: Chad M
authors.initials: CM
authors.lastname: Rienstra
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D003903:Deuterium; D029968:Escherichia coli Proteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry; D011522:Protons; D051844:alpha-Synuclein / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; C079503:DsbB protein, Bacteria; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011522:Protons; D051844:alpha-Synuclein; D003903:Deuterium; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli