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

Abstract

We have developed a methodology for identifying further thermostabilizing mutations for an intrinsically thermostable membrane protein. The methodology comprises the following steps: (1) identifying thermostabilizing single mutations (TSSMs) for residues in the transmembrane region using our physics-based method; (2) identifying TSSMs for residues in the extracellular and intracellular regions, which are in aqueous environment, using an empirical force field FoldX; and (3) combining the TSSMs identified in steps (1) and (2) to construct multiple mutations. The methodology is illustrated for thermophilic rhodopsin whose apparent midpoint temperature of thermal denaturation T m is ∼91.8 °C. The TSSMs previously identified in step (1) were F90K, F90R, and Y91I with ΔT m ∼5.6, ∼5.5, and ∼2.9 °C, respectively, and those in step (2) were V79K, T114D, A115P, and A116E with ΔT m ∼2.7, ∼4.2, ∼2.6, and ∼2.3 °C, respectively (ΔT m denotes the increase in T m). In this study, we construct triple and quadruple mutants, F90K+Y91I+T114D and F90K+Y91I+V79K+T114D. The values of ΔT m for these multiple mutants are ∼11.4 and ∼13.5 °C, respectively. T m of the quadruple mutant (∼105.3 °C) establishes a new record in a class of outward proton pumping rhodopsins. It is higher than T m of Rubrobacter xylanophilus rhodopsin (∼100.8 °C) that was the most thermostable in the class before this study.

A methodology for creating mutants of G‐protein coupled receptors stabilized in active state by combining statistical thermodynamics and evolutionary molecular engineering

“…The free‐energy function defined in our statistical‐thermodynamics theory 7,8,20–26 comprises entropic and energetic components, but the key residues are searched out using the entropic component. We define −ΔΔ S / k B as $$$$ -\mathsf{\varDelta \varDelta}\mathit{\mathsf{S}}/{\mathit{\mathsf{k}}}_{\mathsf{B}}=-\mathsf{\varDelta}\mathit{\mathsf{S}}/{\mathit{\mathsf{k}}}_{\mathsf{B}}\left(\mathsf{Mutant}\right)-\left\{-\mathsf{\varDelta}\mathit{\mathsf{S}}/{\mathit{\mathsf{k}}}_{\mathsf{B}}\left(\mathsf{WT}\right)\right\} $$$$ where Δ S (Δ S > 0) is the gain of translational, configurational entropy of the solvent upon GPCR folding and k B is the Boltzmann constant.…”

“…The solvent is formed by the hydrocarbon (CH 2 , CH 3 , and CH) groups constituting nonpolar chains of lipid molecules in the TM region. 7,8,[20][21][22][23][24][25][26] ΔS arises primarily from the close packing of side chains accompanying an increase in the total volume available for translational displacement of the hydrocarbon groups.…”

“…(Consult Chap. 1 of Supplementary Material, the first article, 3 and our earlier publications 7,8,[20][21][22][23][24][25][26] for more detailed explanations on the physical meaning of ΔS and the procedure of calculating ΔS.) ÀΔΔS/k B < 0 implies that the mutant is more stable than the WT in terms of the solvent entropy.…”

Rhodopsins: An Excitingly Versatile Protein Species for Research, Development and Creative Engineering

“…With only 35% sequence identity, the crystal structures are remarkably similar, but the gating residues for Cl − and Na + are located at the opposite site of the membrane ( Kato et al, 2015a ; Hosaka et al, 2016 ; Kovalev et al, 2019 ; Yun et al, 2020 ). Another example is the huge mutagenesis effort that converted a thermophilic rhodopsin into the best thermally stable rhodopsin available to date, while retaining pump activity ( Yasuda et al, 2022 ). On the other hand, selective mutations in the opsin could convert BR into an inward chloride pump, the sodium pump KR2 into a selective light-driven cation channel, the proton pumps Archaerhodopsin-3 (AR3) and Coccomyxa subellipsoidea rhodopsin (CsR) into light-driven proton channels, and the proton pump GR from Gloeobacter violaceus into a fluorescent chloride sensor ( Sasaki et al, 1995 ; Brown et al, 1996 ; Inoue et al, 2015 ; 2016 ; Fudim et al, 2019 ; Vogt et al, 2019 ; Tutol et al, 2021 ).…”

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

“…ΔS is a solvent-entropy gain originating from an increase in the total volume available for the translational displacement of solvent particles (i.e., the hydrocarbon groups referred to above) (see Section 4.1 for more details). In our earlier studies, 10,11,[15][16][17][18][19][20][21] it was shown that ΔF/(k B T 0 ) evaluated at T = T 0 is a good measure of the GPCR thermostability. Formation of more intramolecular hydrogen bonds and closer packing of side chains lead to lower values of ΔΛ and ÀTΔS, respectively (see Sections 4.1 and 4.2).…”