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A photon-free approach to transmembrane protein structure determination.

ABSTRACT: The structures of membrane proteins are generally solved using samples dissolved in micelles, bicelles, or occasionally phospholipid bilayers using X-ray diffraction or magnetic resonance. Because these are less than perfect mimics of true biological membranes, the structures are often confirmed by evaluating the effects of mutations on the properties of the protein in their native cellular environments. Low-resolution structures are also sometimes generated from the results of site-directed mutagenesis when other structural data are incomplete or not available. Here, we describe a rapid and automated approach to determine structures from data on site-directed mutants for the special case of homo-oligomeric helical bundles. The method uses as input an experimental profile of the effects of mutations on some property of the protein. This profile is then interpreted by assuming that positions that have large effects on structure/function when mutated project toward the center of the oligomeric bundle. Model bundles are generated, and correlation analysis is used to score which structures have inter-subunit C(?) distances between adjoining monomers that best correlate with the experimental profile. These structures are then clustered and refined using energy-based minimization methods. For a set of 10 homo-oligomeric TM protein structures ranging from dimers to pentamers, we show that our method predicts structures to within 1-2 Å backbone RMSD relative to X-ray and NMR structures. This level of agreement approaches the precision of NMR structures solved in different membrane mimetics.

SUBMITTER: Soto CS

PROVIDER: S-EPMC3808161 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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A photon-free approach to transmembrane protein structure determination.

Soto Cinque S CS Hannigan Brett T BT DeGrado William F WF

Journal of molecular biology 20111015 4

The structures of membrane proteins are generally solved using samples dissolved in micelles, bicelles, or occasionally phospholipid bilayers using X-ray diffraction or magnetic resonance. Because these are less than perfect mimics of true biological membranes, the structures are often confirmed by evaluating the effects of mutations on the properties of the protein in their native cellular environments. Low-resolution structures are also sometimes generated from the results of site-directed mut ...[more]

PMID: 22024595

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Project description:The two principal components of biological membranes, the lipid bilayer and the proteins integrated within it, have coevolved for specific functions that mediate the interactions of cells with their environment. Molecular structures can provide very significant insights about protein function. In the case of membrane proteins, the physical and chemical properties of lipids and proteins are highly interdependent; therefore structure determination should include the membrane environment. Considering the membrane alongside the protein eliminates the possibility that crystal contacts or detergent molecules could distort protein structure, dynamics, and function and enables ligand binding studies to be performed in a natural setting. Solid-state NMR spectroscopy is compatible with three-dimensional structure determination of membrane proteins in phospholipid bilayer membranes under physiological conditions and has played an important role in elucidating the physical and chemical properties of biological membranes, providing key information about the structure and dynamics of the phospholipid components. Recently, developments in the recombinant expression of membrane proteins, sample preparation, pulse sequences for high-resolution spectroscopy, radio frequency probes, high-field magnets, and computational methods have enabled a number of membrane protein structures to be determined in lipid bilayer membranes. In this Account, we illustrate solid-state NMR methods with examples from two bacterial outer membrane proteins (OmpX and Ail) that form integral membrane ?-barrels. The ability to measure orientation-dependent frequencies in the solid-state NMR spectra of membrane-embedded proteins provides the foundation for a powerful approach to structure determination based primarily on orientation restraints. Orientation restraints are particularly useful for NMR structural studies of membrane proteins because they provide information about both three-dimensional structure and the orientation of the protein within the membrane. When combined with dihedral angle restraints derived from analysis of isotropic chemical shifts, molecular fragment replacement, and de novo structure prediction, orientation restraints can yield high-quality three-dimensional structures with few or no distance restraints. Using complementary solid-state NMR methods based on oriented sample (OS) and magic angle spinning (MAS) approaches, one can resolve and assign multiple peaks through the use of (15)N/(13)C labeled samples and measure precise restraints to determine structures.

Dataset Information

A photon-free approach to transmembrane protein structure determination.

Publications

A photon-free approach to transmembrane protein structure determination.

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