A photon-free approach to transmembrane protein structure determination.
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(β) 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.
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. css38@columbia.edu
Cinque S
CS
Soto
Brett T
BT
Hannigan
William F
WF
DeGrado
D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017785:Photons; D017433:Protein Structure, Secondary; D013223:Statistics as Topic; D014961:X-Ray Diffraction / Q000379:methods
D008565:Membrane Proteins