Equilibrium and Non-equilibrium Studies of Protein G using High-Resolution Optical Trapping Unveil Heterogeneous Unfolding Pathways

Abstract

solution measurements, and the challenges in incorporating them into computational prediction. As improvement opportunities were only partly realized in CASP12, we provide guidance on how data from the full-length biological unit and the solution state can better aid prediction of the folded monomer or subunit. We furthermore describe strategic integrations of solution measurements with computational prediction programs with the aim of substantially improving foundational knowledge and the accuracy of computational algorithms for biologically-relevant structure predictions for proteins in solution. 2856-Pos Board B64Mimicking Microbial Rhodopsin Isomerization Photoreceptor proteins, like microbial rhodopsins, play crucial roles in sensing and responding to light. The microbial rhodopsins all contain a retinylidene chromophore bound via Schiff base to a lysine. 1 Upon absorption of light, retinal isomerizes from all-trans to 13-cis, driving a protein conformational change; then the all-trans retinal is rapidly and thermally regenerated at the end of the photocycle. The rhodopsin must: channel the photo-isomerization to the C13-C14 double bond exclusively; overcome the energy barrier for photo-isomerization; and induce the rapid, thermal return to its original all trans form. 1,2 Research investigating this isomerization pathway has involved either chemical modification or mutational studies. An alternative approach would be to engineer a system from scratch that recapitulates all of the functions of a microbial rhodopsin, which would result in a much deeper understanding of the protein/chromophore interactions required to make a rhodopsin a reality. Herein we report just such a system using human Cellular Retinoic Acid Binding Protein II (hCRABPII) as a template. In this system, the photo-isomerization of retinal from all-trans to 13-cis 15-syn was demonstrated both in solution and in protein crystals, where atomic resolution structures after photo-isomerization showed both quantitative and specific photo-isomerization of the 13-bond and subsequent thermal relaxation exclusively to the all-trans isomer, fully recapitulating the microbial rhodopsin photocycle. In addition, the crystal structure of a 13-cis retinal-bound protein complex was determined at atomic resolution for the first time, showing how proteins can stabilize this isomeric form of retinal. 1-Ernst, O. et al.