Identification of thermostabilizing mutations for a membrane protein whose three‐dimensional structure is unknown

We recently developed a physics‐based method for identifying thermostabilizing mutations of a membrane protein. The method uses a free‐energy function F where the importance of translational entropy of hydrocarbon groups within the lipid bilayer is emphasized. All of the possible mutations can rapidly be examined. The method was illustrated for the adenosine A2a receptor (A2aR) whose three‐dimensional (3D) structure experimentally determined was utilized as the wild‐type structure. Nine single mutations and a double mutation predicted to be stabilizing or destabilizing were checked by referring to the experimental results: The success rate was remarkably high. In this work, we postulate that the 3D structure of A2aR is unknown. We construct candidate models for the 3D structure using the homology modeling and select the model giving the lowest value to the change in F on protein folding. The performance achieved is only slightly lower than that in the recent work. © 2016 Wiley Periodicals, Inc.