Dataset Information

Triton X-100 as the "short-chain lipid" improves the magnetic alignment and stability of membrane proteins in phosphatidylcholine bilayers for oriented-sample solid-state NMR spectroscopy.

ABSTRACT: A mixture of phospholipids and Triton X-100 in a molar ratio of 5:1 forms well-aligned and stable bilayers that give superior solid-state NMR spectra of proteins. In a comparison, the oriented-sample (OS) solid-state NMR spectrum of Pf1 coat protein in aligned phospholipid bilayers displayed better resolution than the equivalent solution NMR spectrum of the same protein in micelles. Both samples and experimental parameters were fully optimized.

SUBMITTER: Park SH

PROVIDER: S-EPMC2958821 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Triton X-100 as the "short-chain lipid" improves the magnetic alignment and stability of membrane proteins in phosphatidylcholine bilayers for oriented-sample solid-state NMR spectroscopy.

Park Sang Ho SH Opella Stanley J SJ

Journal of the American Chemical Society 20100901 36

A mixture of phospholipids and Triton X-100 in a molar ratio of 5:1 forms well-aligned and stable bilayers that give superior solid-state NMR spectra of proteins. In a comparison, the oriented-sample (OS) solid-state NMR spectrum of Pf1 coat protein in aligned phospholipid bilayers displayed better resolution than the equivalent solution NMR spectrum of the same protein in micelles. Both samples and experimental parameters were fully optimized. ...[more]

PMID: 20735058

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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

Triton X-100 as the "short-chain lipid" improves the magnetic alignment and stability of membrane proteins in phosphatidylcholine bilayers for oriented-sample solid-state NMR spectroscopy.

Publications

Triton X-100 as the "short-chain lipid" improves the magnetic alignment and stability of membrane proteins in phosphatidylcholine bilayers for oriented-sample solid-state NMR spectroscopy.

Similar Datasets

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