"Use of Fluorescence Resonance Energy Transfer for Detection of Lattice Formation in ASR"
Anabaena sensory rhodopsin (ASR) is a microbial sensory rhodopsin which was found in freshwater cyanobacterium Anabaena sp. PCC 7120. ASR is known to form trimers in lipids and detergent. Furthermore, recent SAXS results suggested that the trimers assemble into the BR-like hexagonal lattice (a 2D crystal). This project aimed to investigate the hexagonal lattice formation of ASR by developing and applying Fluorescence Resonance Energy Transfer (FRET) approach to this biological problem. When the hexagonal lattice formation occurs the distances between monomers in different trimers can be even shorter than distances between monomers of the same trimer, resulting in strong intertrimer FRET signals. To observe the intertrimer FRET, mutant S26C ASR was successfully labelled with dyes constituting a FRET pair, Alexa Fluor 555 and Alexa Fluor 647, at non-native cysteine C26, and other dye pairs were tested as well. The conditions for successful selective labeling of ASR were found, a strong FRET was detected, and the effects of environmental factors such as 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 (but not a complete lattice dissolution), while the fluidity of lipids had no significant effect on the intermolecular distances.
Dr. Hermann Eberl, Chair (Department of Mathematics and Statistics)
Dr. Leonid Brown, Advisor (Department of Physics)
Dr. Vladimir Ladizhansky, Advisory Committee (Department of Physics)