How Does a Microbial Rhodopsin RxR Realize Its Exceptionally High Thermostability with the Proton-Pumping Function Being Retained?

Abstract

We often encounter a case where two proteins, whose amino-acid sequences are similar, are quite different with regard to the thermostability. As a striking example, we consider the two seven-transmembrane proteins: recently discovered Rubrobacter xylanophilus rhodopsin (RxR) and long-known bacteriorhodopsin from Halobacterium salinarum (HsBR). They commonly function as a light-driven proton pump across the membrane. Though their sequence similarity and identity are ∼71 and ∼45%, respectively, RxR is much more thermostable than HsBR. In this study, we solve the three-dimensional structure of RxR using X-ray crystallography and find that the backbone structures of RxR and HsBR are surprisingly similar to each other: The root-mean-square deviation for the two structures calculated using the backbone Cα atoms of the seven helices is only 0.86 Å, which makes the large stability difference more puzzling. We calculate the thermostability measure and its energetic and entropic components for RxR and HsBR using our recently developed statistical-mechanical theory. The same type of calculation is independently performed for the portions playing essential roles in the proton-pumping function, helices 3 and 7, and their structural properties are related to the probable roles of water molecules in the proton-transporting mechanism. We succeed in elucidating how RxR realizes its exceptionally high stability with the original function being retained. This study provides an important first step toward the establishment of a method correlating microscopic, geometric characteristics of a protein with its thermodynamic properties and enhancing the thermostability through amino-acid mutations without vitiating the original function.

Applicability of Styrene-Maleic Acid Copolymer for Two Microbial Rhodopsins, RxR and HsSRI

“…Because the average diameter of the particles was estimated as 30 5 6.4 nm, the average number of lipids contained in an empty particle with a diameter of 30 nm was estimated as 1125$1250 lipids, assuming that an empty particle with a diameter of 12 nm contains 180$200 lipids (35). The volume of 4.1 trimers of RxR corresponds to 492 lipids, assuming that the volume of crystal structure of monomeric RxR ($37 nm 3 ) corresponds to 40 lipids (42). By subtracting 492 lipids from 1125 to $1250 lipids, we calculated the number of lipids as 635$760 in the particle with a diameter of 30 nm containing 4.1 timers of RxR.…”

A fully automated crystallization apparatus for small protein quantities

“…The original PXS set up 676 plates per year on average (from 2003 to 2016) and PXS2 set up 894 plates per year (from 2017 to mid-2020), including membrane proteins. The outcome of PXS2 is beginning to become apparent (Hayashi et al, 2020;Koiwai et al, 2020;Kuwabara et al, 2020). Our PXS2 system can be accessed through the BINDS (Basis for Supporting Innovative Drug Discovery and Life Science Research) project, which is a Japanese national research project to establish an innovative platform for expediting the therapeutic applications of early-stage drugdiscovery and medical technology advances.…”

“…To confirm the performance of PXS2 with membrane proteins, we used two test samples, Neisseria meningitidis PorB as a -barrel protein (Tanabe et al, 2010) and Rubrobacter xylanophilus rhodopsin (RxR) as an -helical protein (Hayashi et al, 2020). Concentrated PorB ($15 mg ml À1 ) and RxR ($11 mg ml À1 ) were used to test the improved system.…”

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

“…The crystal structure of the highly thermophilic rhodopsin (TR) from Thermus thermophilus was resolved to be very similar to that of the much less thermally stable xanthorhodopsin (XR) from Salinibacter ruber , including the binding crevice for the carotenoid antenna ( Tsukamoto et al, 2016 ). Likewise, the crystal structure of the thermostable rhodopsin proton-pump from Rubrobacter xylanophilus (RxR) is very similar to that of bacteriorhodopsin ( Hayashi et al, 2020 ). An unusually widely stable proton pump (pH, detergent, temperature), named Tara76 rhodopsin, was isolated from uncultured bacteria ( Shim et al, 2021 ).…”

“…An unusually widely stable proton pump (pH, detergent, temperature), named Tara76 rhodopsin, was isolated from uncultured bacteria ( Shim et al, 2021 ). Such data shed new light on the design options to increase thermal and environmental stability without a significant sacrifice in dynamics and activity ( Hayashi et al, 2020 ).…”