Over the past few
decades, the structure, functions, properties,
and molecular mechanisms of retinal proteins have been studied extensively.
The newly studied retinal protein Gloeobacter rhodopsin (gR) acts
as a light-driven proton pump, transferring a proton from the cytoplasmic
region to the extracellular region of a cell following light absorption.
It was previously shown that gR can bind the carotenoid salinixanthin
(sal). In the present study, we report the effect of pH on the binding
of retinal to the apo-protein of gR, in the presence and absence of
sal, to form the gR pigment. We found that binding at different pH
levels reflects the titration of two different protein residues, one
at the lower p<i>K</i><sub>a</sub> 3.5 and another at the
higher p<i>K</i><sub>a</sub> 8.4, that affect the pigment’s
formation. The maximum amount of pigment was formed at pH 5, both
with and without the presence of sal. The introduction of sal accelerates
the rate of pigment formation by a factor of 190. Furthermore, it
is suggested that occupation of the binding site by the retinal chromophore
induces protein conformational alterations which in turn affect the
carotenoid conformation, which precedes the formation of the retinal–protein
covalent bond. Our examination of synthetic retinal analogues in which
the ring structure was modified revealed that, in the absence of sal,
the retinal ring structure affects the rate of pigment formation and
that the intact structure is needed for efficient pigment formation.
However, the presence of sal abolishes this effect, and all-trans
retinal and its modified ring analogues bind at a similar rate
