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.

Human muscarinic receptor, M 2 is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype selective ligands against one of the five muscarinic receptors. We report high resolution structures of a thermostabilized mutant M 2 receptor bound to a subtype selective antagonist AF-DX 384 and a non-selective antagonist NMS. The thermostabilizing mutation S110R in M 2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M 2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, that is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular Dynamics simulations reveal that tightening of the ligand-residue contacts in M 2 receptor compared to M 3 receptor leads to subtype selectivity of AF-DX 384.

A protein folds into its native structure with the α-helix and∕or β-sheet in aqueous solution under the physiological condition. The relative content of these secondary structures largely varies from protein to protein. However, such structural variability is not exhibited in nonaqueous environment. For example, there is a strong trend that alcohol induces a protein to form α-helices, and many of the membrane proteins within the lipid bilayer consists of α-helices. Here we investigate the structural stability of proteins in aqueous and nonpolar environments using our recently developed free-energy function F = (Λ - TS)∕(k(B)T(0)) = Λ∕(k(B)T(0)) - S∕k(B) (T(0) = 298 K and the absolute temperature T is set at T(0)) which is based on statistical thermodynamics. Λ∕(k(B)T(0)) and S∕k(B) are the energetic and entropic components, respectively, and k(B) is Boltzmann's constant. A smaller value of the positive quantity, -S, represents higher efficiency of the backbone and side-chain packing promoted by the entropic effect arising from the translational displacement of solvent molecules or the CH(2), CH(3), and CH groups which constitute nonpolar chains of lipid molecules. As for Λ, in aqueous solution, a transition to a more compact structure of a protein accompanies the break of protein-solvent hydrogen bonds: As the number of donors and acceptors buried without protein intramolecular hydrogen bonding increases, Λ becomes higher. In nonpolar solvent, lower Λ simply implies more intramolecular hydrogen bonds formed. We find the following. The α-helix and β-sheet are advantageous with respect to -S as well as Λ and to be formed as much as possible. In aqueous solution, the solvent-entropy effect on the structural stability is so strong that the close packing of side chains is dominantly important, and the α-helix and β-sheet contents are judiciously adjusted to accomplish it. In nonpolar solvent, the solvent-entropy effect is substantially weaker than in aqueous solution. Λ is crucial and the α-helix is more stable than the β-sheet in terms of Λ, which develops a tendency that α-helices are exclusively chosen. For a membrane protein, α-helices are stabilized as fundamental structural units for the same reason, but their arrangement is performed through the entropic effect mentioned above.