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Helix conformations in 7TM membrane proteins determined using oriented-sample solid-state NMR with multiple residue-specific 15N labeling.

ABSTRACT: Oriented solid-state NMR in combination with multiple-residue-specific (15)N labeling and extensive numerical spectral analysis is proposed to determine helix conformations of large membrane proteins in native membranes. The method is demonstrated on uniaxially oriented samples of (15)N-methionine, -valine, and -glycine-labeled bacteriorhopsin in native purple membranes. Experimental two-dimensional (1)H-(15)N dipole-dipole coupling versus (15)N chemical shift spectra for all samples are analyzed numerically to establish combined constraints on the orientation of the seven transmembrane helices relative to the membrane bilayer normal. Since the method does not depend on specific resonance assignments and proves robust toward nonidealities in the sample alignment, it may be generally feasible for the study of conformational arrangement and function-induced conformation changes of large integral membrane proteins.

SUBMITTER: Vosegaard T

PROVIDER: S-EPMC2134869 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Helix conformations in 7TM membrane proteins determined using oriented-sample solid-state NMR with multiple residue-specific 15N labeling.

Vosegaard Thomas T Kamihira-Ishijima Miya M Watts Anthony A Nielsen Niels Chr NC

Biophysical journal 20070907 1

Oriented solid-state NMR in combination with multiple-residue-specific (15)N labeling and extensive numerical spectral analysis is proposed to determine helix conformations of large membrane proteins in native membranes. The method is demonstrated on uniaxially oriented samples of (15)N-methionine, -valine, and -glycine-labeled bacteriorhopsin in native purple membranes. Experimental two-dimensional (1)H-(15)N dipole-dipole coupling versus (15)N chemical shift spectra for all samples are analyze ...[more]

PMID: 17827220

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Project description:The validation of protein structures through functional assays has been the norm for many years. Functional assays perform this validation for water-soluble proteins very well, but they need to be performed in the same environment as that used for the structural analysis. This is difficult for membrane proteins that are often structurally characterized in detergent environments, although functional assays for these proteins are most frequently performed in lipid bilayers. Because the structure of membrane proteins is known to be sensitive to the membrane mimetic environment, such functional assays are appropriate for validating the protein construct, but not the membrane protein structure. Here, we compare oriented sample solid-state NMR spectral data of diacylglycerol kinase previously published with predictions of such data from recent structures of this protein. A solution NMR structure of diacylglycerol kinase has been obtained in detergent micelles and three crystal structures have been obtained in a monoolein cubic phase. All of the structures are trimeric with each monomer having three transmembrane and one amphipathic helices. However, the solution NMR structure shows typical perturbations induced by a micelle environment that is reflected in the predicted solid-state NMR resonances from the structural coordinates. The crystal structures show few such perturbations, especially for the wild-type structure and especially for the monomers that do not have significant crystal contacts. For these monomers the predicted and observed data are nearly identical. The thermostabilized constructs do show more perturbations, especially the A41C mutation that introduces a hydrophilic residue into what would be the middle of the lipid bilayer inducing additional hydrogen bonding between trimers. These results demonstrate a general technique for validating membrane protein structures with minimal data obtained from membrane proteins in liquid crystalline lipid bilayers by oriented sample solid-state NMR.

Dataset Information

Helix conformations in 7TM membrane proteins determined using oriented-sample solid-state NMR with multiple residue-specific 15N labeling.

Publications

Helix conformations in 7TM membrane proteins determined using oriented-sample solid-state NMR with multiple residue-specific 15N labeling.

Similar Datasets

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