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

Area of Science:

• Structural biology
• Biophysics
• Protein engineering

Background:

• Proteins with similar amino acid sequences can exhibit significant differences in thermostability.
• Seven-transmembrane proteins like Rubrobacter xylanophilus rhodopsin (RxR) and Halobacterium salinarum bacteriorhodopsin (HsBR) function as proton pumps but differ in stability.
• RxR shows much higher thermostability than HsBR, despite sequence similarities of ~71% and identity of ~45%.

Purpose of the Study:

• To elucidate the structural and energetic basis for the exceptional thermostability of RxR compared to HsBR.
• To investigate how RxR maintains its proton-pumping function while achieving high stability.
• To establish a method for correlating protein structure with thermodynamic properties and enhancing thermostability.

Main Methods:

• X-ray crystallography was used to determine the three-dimensional structure of RxR.
• A statistical-mechanical theory was applied to calculate thermostability measures, including energetic and entropic components, for both RxR and HsBR.
• Analysis focused on key functional regions, specifically helices 3 and 7, and the potential role of water molecules in proton transport.

Main Results:

• The backbone structures of RxR and HsBR are highly similar, with a root-mean-square deviation of 0.86 Å for Cα atoms of the seven helices.
• Thermodynamic calculations revealed the energetic and entropic contributions to the differing thermostability of RxR and HsBR.
• The study identified mechanisms by which RxR achieves high stability without compromising its proton-pumping function.

Conclusions:

• RxR's high thermostability is achieved through specific structural and energetic features, distinct from HsBR.
• Understanding these features provides insights into the role of water molecules in the proton transport mechanism.
• This work is a foundational step towards engineering enhanced protein thermostability through targeted amino acid mutations.