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A microscale protein NMR sample screening pipeline.

ABSTRACT: As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30-200 microg in 8-35 microl volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure.

SUBMITTER: Rossi P

PROVIDER: S-EPMC2797623 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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A microscale protein NMR sample screening pipeline.

Rossi Paolo P Swapna G V T GV Huang Yuanpeng J YJ Aramini James M JM Anklin Clemens C Conover Kenith K Hamilton Keith K Xiao Rong R Acton Thomas B TB Ertekin Asli A Everett John K JK Montelione Gaetano T GT

Journal of biomolecular NMR 20091114 1

As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30-200 microg in 8-3 ...[more]

PMID: 19915800

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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 microscale protein NMR sample screening pipeline.

Publications

A microscale protein NMR sample screening pipeline.

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