Title

Optimization of bacteriorhodopsin via mutagenesis

Date of Completion

January 2004

Keywords

Biology, Molecular|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Bacteriorhodopsin (BR) is a membrane protein found in the cell membrane of the halophilic archaeon Halobacterium salinarum, where the protein functions as a light-driven proton pump under conditions of high light intensity and low oxygen availability. In order to understand the structure and function of BR in greater detail, site-directed and semi-random mutagenesis were used to modify amino acids spanning 96% of the protein. Over 900 site-directed and semi-random mutants were constructed and studied using time-resolved UV-Vis spectroscopy. Mutants that possessed greatly altered photocycle intermediate lifetimes and yields were selected for further scrutiny. Two intermediates in the photocycle of BR were of particular focus: the M state (λ max = 412 nm) and the O state (λmax = 640 nm). In addition to providing important knowledge on the function of BR, mutants were also examined with regard to potential worth in bioelectronic devices, specifically holographic and three-dimensional memories. One sextuple mutant was discovered in a screen of semi-random mutants that possessed an unusually long O state lifetime. This mutant was used to examine residues affecting the O state and the chromophore composition of the O state. This work endorses the use of semi-random and random mutagenesis techniques to detect photophysical mutants for research and applied investigation. ^