Date of Original Version

11-23-1981

Type

Dissertation

Abstract or Description

This study has used chemical modification techniques to

investigate the role of the only two types of positively charged

amino acids, lysines and arginines, in the membrane-bound protein,

bacteriorhodopsin (bR). The results of these chemical modifications

have significance both for the structure of bR and for the molecular

mechanism of light-activated photocycling and proton pumping.

The implications for the secondary structure of bR are:

LYS 40 is totally exposed to the aqueous phase, while the other

five reactive lysines are partially buried in the hydrophobic domain;

LYS 30, LYS 129 and LYS 159 are buried by only one or two residues

within the hydrophobic domain; all but two arginines are totally

exposed to the aqueous phase; and at least three ionic bridges form

between arginines and carboxyl groups. These results were used to

construct a new model of the secondary structure of bR. The implication

for the tertiary structure of bR is that Model A (126) is

the preferred model of fitting the bR sequence into the helices seen

by electron diffraction. The implication for the quaternary structure

is that one structural role of retinal is to increase the intermolecular

distances between bR molecules in a trimer. This focuses

attention on the bR trimer as the primary structural unit.

Regarding the molecular mechanism of photocycling and proton

pumping, two specific arginines were responsible for the slowdown of

the photocycle. This result was obtained by a quantitative analysis

of the inhibition of M decay as a function of fraction of arginines

modified by 2,3-butanedione. Possible roles of the two important

arginines, including controlling the conformational changes of the

protein by ionic linkages to carboxyl groups and participating directly

in proton pathways, are discussed. Bifunctional imidoester modification

of lysines revealed that a conformational change of the bR protein is

needed for photocycling, since cross-linking lysines slowed the photocycle.

2,3-Butanedione modification of arginines supported this

result, since this modification caused a marked slowdown of the photocycle

and also protein conformational changes as evidenced by

tryptophan fluorescence and circular dichroism spectroscopy.

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