No abstract

Membrane proteins are responsible for the communication between cells and their environments. They are indispensable to the expression of life phenomena and also implicated in a number of diseases. Nevertheless, the studies on membrane proteins are far behind those on water-soluble proteins, primarily due to their low structural stability. Introduction of mutations can enhance their thermostability and stability in detergents, but the stabilizing mutations are currently identified by experiments. The recently reported computational methods suffer such drawbacks as the exploration of only limited mutational space and the empiricism whose results are difficult to physically interpret. Here we develop a rapid method that allows us to treat all of the possible mutations. It employs a free-energy function (FEF) that takes into account the translational entropy of hydrocarbon groups within the lipid bilayer as well as the protein intramolecular hydrogen bonding. The method is illustrated for the adenosine A2a receptor whose wild-type structure is known and utilized. We propose a reliable strategy of finding key residues to be mutated and selecting their mutations, which will lead to considerably higher stability. Representative single mutants predicted to be stabilizing or destabilizing were experimentally examined and the success rate was found to be remarkably high. The melting temperature Tm for two of them was substantially higher than that of the wild type. A double mutant with even higher Tm was also obtained. Our FEF captures the essential physics of the stability changes upon mutations.

Although the two membrane proteins, thermophilic rhodopsin (TR) and xanthorhodopsin (XR), share a high similarity in amino-acid sequence and an almost indistinguishable three-dimensional structure, TR is much more thermostable than XR. This is counterintuitive also because TR possesses only a smaller number of intramolecular hydrogen bonds (HBs) than XR. Here we investigate physical origins of the remarkable difference between XR and TR in the stability. Our free-energy function (FEF) is improved so that not only the portion within the transmembrane (TM) region but also the extracellular and intracellular portions within the water-immersed (WI) regions can be considered in assessing the stability. The assessment is performed on the basis of the FEF change upon protein folding, which is calculated for the crystal structure of XR or TR. Since the energetics within the TM region is substantially different from that within the WI regions, we determine the TM and WI portions of XR or TR by analyzing the distribution of water molecules using all-atom molecular dynamics simulations. The energetic component of the FEF change consists of a decrease in energy arising from the formation of intramolecular HBs and an increase in energy caused by the break of protein-water HBs referred to as “energetic dehydration penalty.” The entropic component is a gain of the translational, configurational entropies of hydrocarbon groups within the lipid bilayer and of water molecules. The entropic component is calculated using the integral equation theory combined with our morphometric approach. The energetic one is estimated by a simple but physically reasonable method. We show that TR is much more stable than XR for the following reasons: The decrease in energy within the TM region is larger, and the energetic dehydration penalty within the WI regions is smaller, leading to higher energetic stabilization, and tighter packing of side chains accompanying the association of seven helices confers higher entropic stabilization on TR.

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 A receptor (A R) 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 A R 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.