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A theoretical assessment of structure determination of multi-span membrane proteins by oriented sample solid-state NMR spectroscopy.

ABSTRACT: Oriented sample solid state NMR (OS-ssNMR) spectroscopy allows direct determination of the structure and topology of membrane proteins reconstituted into aligned lipid bilayers. While OS-ssNMR theoretically has no upper size limit, its application to multi-span membrane proteins has not been established since most studies have been restricted to single or dual span proteins and peptides. Here, we present a critical assessment of the application of this method to multi-span membrane proteins. We used molecular dynamics simulations to back-calculate [15N-1H] separated local field (SLF) spectra from a G protein-coupled receptor (GPCR) and show that fully resolved spectra can be obtained theoretically for a multi-span membrane protein with currently achievable resonance linewidths.

SUBMITTER: Weber DK

PROVIDER: S-EPMC7643874 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A theoretical assessment of structure determination of multi-span membrane proteins by oriented sample solid-state NMR spectroscopy.

Weber Daniel K DK Veglia Gianluigi G

Australian journal of chemistry 20190920 3

Oriented sample solid state NMR (OS-ssNMR) spectroscopy allows direct determination of the structure and topology of membrane proteins reconstituted into aligned lipid bilayers. While OS-ssNMR theoretically has no upper size limit, its application to multi-span membrane proteins has not been established since most studies have been restricted to single or dual span proteins and peptides. Here, we present a critical assessment of the application of this method to multi-span membrane proteins. We ...[more]

PMID: 33162560

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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

A theoretical assessment of structure determination of multi-span membrane proteins by oriented sample solid-state NMR spectroscopy.

Publications

A theoretical assessment of structure determination of multi-span membrane proteins by oriented sample solid-state NMR spectroscopy.

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