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

Abstract

It is known that a hyperthermostable protein tolerable at temperatures over 100°C can be designed from a soluble globular protein by introducing mutations. To expand the applicability of this technology to membrane proteins, here we report a further thermo‐stabilization of the thermophilic rhodopsin from Thermus thermophilus JL‐18 as a model membrane protein. Ten single mutations in the extramembrane regions were designed based on a computational prediction of folding free‐energy differences upon mutation. Experimental characterizations using the UV‐visible spectroscopy and the differential scanning calorimetry revealed that four of ten mutations were thermo‐stabilizing: V79K, T114D, A115P, and A116E. The mutation‐structure relationship of the TR constructs was analyzed using molecular dynamics simulations at 300 K and at 1800 K that aimed simulating structures in the native and in the random‐coil states, respectively. The native‐state simulation exhibited an ion‐pair formation of the stabilizing V79K mutant as it was designed, and suggested a mutation‐induced structural change of the most stabilizing T114D mutant. On the other hand, the random‐coil‐state simulation revealed a higher structural fluctuation of the destabilizing mutant S8D when compared to the wild type, suggesting that the higher entropy in the random‐coil state deteriorated the thermal stability. The present thermo‐stabilization design in the extramembrane regions based on the free‐energy calculation and the subsequent evaluation by the molecular dynamics may be useful to improve the production of membrane proteins for structural studies.

Development of an Outward Proton Pumping Rhodopsin with a New Record in Thermostability by Means of Amino Acid Mutations

“… a These values were reported in our earlier works. , b The number within the parentheses indicates the increase in T m achieved by the mutation. …”

“…However, when the difference between the values of Δ G for a mutant and the WT is taken, the contributions from the residues in the TM region are canceled out. Taken together, in method 2 where FoldX is employed, the prediction is reliable only for a mutation of a residue in the EC or IC region.…”

“…In other words, a mutation which is stabilizing both theoretically and experimentally modifies only the local structure around the mutated residue. On the other hand, a mutation recommended by method 2 is in the loop portion in aqueous environment . The mutation is not likely to significantly modify the seven TM helices.…”

“…In recent works, we succeeded in identifying thermostabilizing mutations for TR using two different methods, methods 1 and 2, with its original function as a proton pump being retained. , In method 1, we developed a theory based on statistical thermodynamics which is different from that developed for GPCRs mentioned above. It is best suited to the further stabilization of a membrane protein whose wild type (WT) is highly thermostable.…”

“…A double mutation F90K+Y91I was also tested, and its Δ T m was ∼8.0 °C. In method 2, where an empirical force field FoldX is employed, by contrast the result is reliable only for a mutation of a residue in the EC or IC portion as explained in “Empirical Method Using FoldX for a Water-Soluble Protein (Method 2)”. Ten mutations were suggested and four of them (V79K, T114D, A115P, and A116E) were actually stabilizing: The success rate was 4/10.…”