Title

Genetic optimization and time-resolved spectroscopy of bacteriorhodopsin variants

Date of Completion

January 2006

Keywords

Chemistry, Analytical|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

The protein bacteriorhodopsin (BR) functions as a light-driven proton pump in its native organism Halobacterium salinarum and can be applied to a number of biophotonic devices. Genetic changes made to BR that affect proton pumping are visualized through spectral changes to the photocycle. Changes that affect the M and O state photokinetics are promising candidates for use in a number of biophotonic devices, The spectrokinetic properties of over 3,000 variant forms of BR, encompassing 96% of the protein structure, are studied by using time-resolved UV/Visible spectroscopy at three wavelengths: 412 nm, 658 nm and 650 nm. Protein variants with altered photocycles are further studied by using time-resolved Fourier transform infrared (FT-IR) spectroscopy and Fourier transform terahertz (FT-THz) spectroscopy. Protein variants are constructed by using site-directed and semi-random mutagenesis techniques. The results endorse the use of semi-random mutagenesis combined with spectroscopic methods to identify protein variants with novel photokinetic properties. ^