Biochemical and Biophysical Characterization of Type I Retinal Binding Proteins for their Application in Device Electronics
Date of Completion
Biology, Molecular|Chemistry, Biochemistry|Biophysics, General
Type I retinal binding proteins are simple photoactive membrane proteins that translocate ions against a chemical gradient to serve a biological function. Harnessing the photophysical properties of two such proton pumps, Bacteriorhodopsin (BR) and Proteorhodopsin (PR), is of interest for application in biophotonic (e.g., volumetric and associative memories) and biomimetic (e.g., artificial retina) technologies. Proteorhodopsin, a recently discovered cubacterial rhodopsin, is found throughout the oceans of the Earth and exhibits many similarities with BR, which has long been the paradigm of protein based device electronics. Many of the current biochemical and biophysical studies of PR use a detergent solubilized form of the protein that is expressed in Escherichia coli. The first half of this thesis characterizes the structural and photochemical properties of PR in non-ionic detergent and various nanoscale phospholipid bilayers, or so-called nanodisc structures. These studies demonstrate that the detergent solubilized protein is both photochemically (9,000 to 70,000 photocycles protein -1) and thermally (>75 °C) stable. Incorporation of PR into small or large nanodiscs demonstrates that PR exists as a photoactive oligomer that exhibits a moderately fast photocycle (∼ 100 ins) in thick phospholipid bilayer. The second half of this thesis characterizes a novel blue BR mutant (i.e., D85E/D96Q) that is capable of forming the Q state photoproduct with >99% efficiency at pH 8.5. The Q photoproduct is the basis of several biophotonic technologies that employ BR as the photoactive element, but have yet to be practical because of the low Q formation efficiency from native and mutant BRs. The D85E/D96Q mutant also exhibits higher synthesis yields of the stable protein and the greatest stability of all investigated blue BR mutants. ^
Ranaghan, Matthew J, "Biochemical and Biophysical Characterization of Type I Retinal Binding Proteins for their Application in Device Electronics" (2011). Doctoral Dissertations. AAI3468087.