Use of Fluorescence Resonance Energy Transfer for Detection of Lattice Formation in ASR

Abstract

Anabaena sensory rhodopsin (ASR) is a microbial sensory rhodopsin which is known to form trimers in lipids and detergent. Furthermore, recent SAXS results suggested that the trimers assemble into the BR-like hexagonal lattice. This project aimed to investigate the hexagonal lattice formation of ASR by developing and applying Fluorescence Resonance Energy Transfer method. When the hexagonal lattice formation occurs the distances between monomers in different trimers will be short, resulting in strong intertrimer FRET signals. Mutant S26C ASR was successfully labelled with FRET pair, Alexa Fluor 555 and Alexa Fluor 647, at non-native cysteine C26 and strong FRET was detected. The effects of environmental factors, packing density and fluidity of lipids, on lattice formation of ASR were investigated. As lipid content was increased, relative FRET efficiency reported a modest increase in the intermolecular distances between monomers in different trimers without complete lattice dissolution, while the fluidity of lipids had no significant effect on the intermolecular distances.