Identification of Thermostabilizing Mutations for Membrane Proteins: Rapid Method Based on Statistical Thermodynamics

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.

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

“…Our free‐energy function is expressed by where k B is the Boltzmann constant, T is the absolute temperature, and T is set at T 0 . Λ (Λ < 0) represents the protein intramolecular energy of the formation of IHBs.…”

“…Taken together, we use a simplified model for the solvent: an ensemble of neutral hard spheres whose diameter and packing fraction are set at those of water at 298 K and 1 atm. The validity of this model was proved in discriminating the native structure from ∼15,000 non‐native structures for GpA …”

“…It is convenient to consider protein folding in terms of the two‐stage model: Individual α‐helices of a membrane protein are separately stabilized as constituent domains within the lipid bilayer in the first stage, and the native structure is completed by the side‐to‐side association of these helices (i.e., association of the transmembrane (TM) portions accompanying the packing of side chains) in the second stage. The solvent‐entropy gain occurring in the second stage is considerably larger than that in the first stage . Hence, Δ S is calculated by focusing on this side‐chain packing (see Fig.…”

“…An important point is that all of the possible mutations (not limited to Ala scanning mutagenesis) are to be examined. We recently developed a theoretical method for identifying thermostabilizing mutations of a membrane protein . The method possesses the following three features: The importance of translational entropy (TE) of hydrocarbon groups within the lipid bilayer is emphasized; all of the possible mutations can rapidly be examined; and when a particular mutation has turned out be thermostabilizing, physical origins of the stabilization can be made clear because the method is a physics‐based one.…”

“…Δ S , for example, represents the gain of TE of hydrocarbon groups on protein folding. Our basic procedure is as follows . First, key residues are nominated using ΔΔ S .…”