Date of Award

1995

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Rosalie K. Crouch

Second Advisor

Daniel R. Knapp

Third Advisor

Kevin J. Schey

Fourth Advisor

Barry Ledford

Fifth Advisor

Erika E. Büllesbach

Abstract

Bacteriorhodopsin (BR) is the sole protein found in the purple membrane of Halobacteria salinarium. The chromophore of BR, all-trans retinal, is bound to the apoprotein of BR via a protonated Schiff base linkage to the E-amino group of Lys216. The protonated all-trans retinal isomerizes to the deprotonated I3-cis retinal upon the absorption of light. This light induced chromophore isomerization triggers a cycle of proton pumping from the cytosol to the extracellular side of the membrane (one H+ per trans to cis isomerization). This proton pumping creates a gradient across the membrane which can be used by the bacterium to synthesize ATP under anaerobic conditions. Although the photocycle and the structure of BR have been studied extensively, the exact distances between the seven α-helices of BR have yet to be totally elucidated. In this thesis, the development of the methodology for using the molecular ruler, 4-(N-maleimido)benzophenone (MBP), to further map interhelical distances of BR is presented. First the parameters of cross-linking with MBP in a model study with the tripeptide glutathione is developed. MBP is found to react with the cysteine residue in glutathione and to photo-insert into C-H bonds as shown by mass spectrometry (MS) and high performance liquid chromatography (HPLC) data. Next the cross-linking methodology is applied to a BR system consisting of a site directed mutant of BR. The maleimide portion of MBP is specific for sulfhydryl groups, however there are no native cysteine residues in BR. A site-directed mutant of BR (T121C) is used which has a Thr replaced by a Cys at position 121. So, MBP will only react with this Cys residue which resides in the hydrophobic interior of BR. The benzophenone portion of MBP can then be photoactivated to a biradical triplet excited state. This excited state will insert into C-H bonds 10 Å from to the maleimide Cys121 bond (the distance between the two functional groups of MBP is 10 Å). Using the Henderson model of BR and molecular modeling, amino acid residues which are possible candidates for photoinsertion by MBP are determined. Experimental data suggests the formation of a MBP-Cys121 bond within T121 C. And the molecular weight of the CNBr digest fragments of the postulated photolysis fragments have been determined by MALDI mass spectrometry. Using the analytical techniques HPLC and MS, the methodology for cross-linking with MBP has been developed. Application of this crosslinking methodology to T121e and BR will be discussed in the text.

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