Methodology for Further Thermostabilization of an Intrinsically Thermostable Membrane Protein Using Amino Acid Mutations with Its Original Function Being Retained

Abstract

We develop a new methodology best suited to the identification of thermostabilizing mutations for an intrinsically stable membrane protein. The recently discovered thermophilic rhodopsin, whose apparent midpoint temperature of thermal denaturation T m is measured to be ∼91.8 °C, is chosen as a paradigmatic target. In the methodology, we first regard the residues whose side chains are missing in the crystal structure of the wild type (WT) as the “residues with disordered side chains,” which make no significant contributions to the stability, unlike the other essential residues. We then undertake mutating each of the residues with disordered side chains to another residue except Ala and Pro, and the resultant mutant structure is constructed by modifying only the local structure around the mutated residue. This construction is based on the postulation that the structure formed by the other essential residues, which is nearly optimized in such a highly stable protein, should not be modified. The stability changes arising from the mutations are then evaluated using our physics-based free-energy function (FEF). We choose the mutations for which the FEF is much lower than for the WT and test them by experiments. We successfully find three mutants that are significantly more stable than the WT. A double mutant whose T m reaches ∼100 °C is also discovered.

A methodology for creating thermostabilized mutants of G‐protein coupled receptors by combining statistical thermodynamics and evolutionary molecular engineering

“…It was previously shown that the mutation S162K is stabilizing, 14 but the present study suggests that the mutation F332K or F332R leads to higher stabilization. Of course, in general, many of the mutations for a membrane protein become stabilizing for different physical reasons and our statistical‐thermodynamics theory is capable of identifying such stabilizing mutations as previously demonstrated 10,15,20 …”

“…Of course, in general, many of the mutations for a membrane protein become stabilizing for different physical reasons and our statistical-thermodynamics theory is capable of identifying such stabilizing mutations as previously demonstrated. 10,15,20 F I G U R E 2 (a) Procedure of fluorescence screening followed. The library prepared was transformed by electroporation.…”

Further thermo‐stabilization of thermophilic rhodopsin from Thermus thermophilusJL‐18 through engineering in extramembrane regions

“…In conclusion, the present thermo‐stabilization design in the extramembrane regions based on the free‐energy calculation and the subsequent evaluation by the MD simulation may be useful to improve the production of membrane proteins. The engineering in the intramembrane region 7,8 . would be a complementary design method.…”

“…Therefore, a rational design of thermostable mutants is a desirable technology in the structural biology of membrane proteins. Recently, a computational method has been developed to identify possible thermo‐stabilizing mutations in the intramembrane region of a membrane protein 7,8 . However, there is no report to date for the rational thermo‐stabilization design in the extramembrane regions.…”

“…Recently, a computational method has been developed to identify possible thermo-stabilizing mutations in the intramembrane region of a membrane protein. 7,8 However, there is no report to date for the rational thermo-stabilization design in the extramembrane regions.…”