title: Identifying inter-residue resonances in crowded 2D (13)C- (13)C chemical shift correlation spectra of membrane proteins by solid-state MAS NMR difference spectroscopy.
abstract: The feasibility of using difference spectroscopy, i.e. subtraction of two correlation spectra at different mixing times, for substantially enhanced resolution in crowded two-dimensional (13)C-(13)C chemical shift correlation spectra is presented. With the analyses of (13)C-(13)C spin diffusion in simple spin systems, difference spectroscopy is proposed to partially separate the spin diffusion resonances of relatively short intra-residue distances from the longer inter-residue distances, leading to a better identification of the inter-residue resonances. Here solid-state magic-angle-spinning NMR spectra of the full length M2 protein embedded in synthetic lipid bilayers have been used to illustrate the resolution enhancement in the difference spectra. The integral membrane M2 protein of Influenza A virus assembles as a tetrameric bundle to form a proton-conducting channel that is activated by low pH and is essential for the viral lifecycle. Based on known amino acid resonance assignments from amino acid specific labeled samples of truncated M2 sequences or from time-consuming 3D experiments of uniformly labeled samples, some inter-residue resonances of the full length M2 protein can be identified in the difference spectra of uniformly (13)C labeled protein that are consistent with the high resolution structure of the M2 (22-62) protein (Sharma et al., Science 330(6003):509-512, 2010).
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, USA.
authors.forename: Yimin
authors.initials: Y
authors.lastname: Miao
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Cross
authors.forename: Riqiang
authors.initials: R
authors.lastname: Fu
mesh_terms: D002247:Carbon Isotopes; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular
chemical_list: D002247:Carbon Isotopes; D008081:Liposomes; D008565:Membrane Proteins