%0 Journal Article
%A Morrow, J. M.
%A S. W. Chang, B.
%D 2015
%T Comparative Mutagenesis Studies of Retinal Release
in Light-Activated Zebrafish Rhodopsin Using Fluorescence Spectroscopy
%U https://acs.figshare.com/articles/journal_contribution/Comparative_Mutagenesis_Studies_of_Retinal_Release_in_Light_Activated_Zebrafish_Rhodopsin_Using_Fluorescence_Spectroscopy/2145961
%R 10.1021/bi501377b.s001
%2 https://acs.figshare.com/ndownloader/files/3779812
%K nonmammalian rhodopsin
%K zebrafish rhodopsin
%K Fluorescence SpectroscopyRhodopsin
%K Schiff base linkage
%K transmembrane helix 5
%K protein moiety
%K EL
%K release rates
%K fluorescence assay
%K extracellular loop 2
%K TM 3
%K model systems
%K Schiff base hydrolysis
%K Arrhenius plot
%K metarhodopsin II
%K Functional variation
%K steric effects
%K Retinal Release
%K Schiff base
%K Comparative Mutagenesis Studies
%K adaptive property
%K activation energy
%K Schiff base stability
%X Rhodopsin is the visual pigment responsible
for initiating scotopic
(dim-light) vision in vetebrates. Once activated by light, release
of all-trans-retinal from rhodopsin involves hydrolysis
of the Schiff base linkage, followed by dissociation of retinal from
the protein moiety. This kinetic process has been well studied in
model systems such as bovine rhodopsin, but not in rhodopsins from
cold-blooded animals, where physiological temperatures can vary considerably.
Here, we characterize the rate of retinal release from light-activated
rhodopsin in an ectotherm, zebrafish (Danio rerio), demonstrating in a fluorescence assay that this process occurs
more than twice as fast as bovine rhodopsin at similar temperatures
in 0.1% dodecyl maltoside. Using site-directed mutagenesis, we found
that differences in retinal release rates can be attributed to a series
of variable residues lining the retinal channel in three key structural
motifs: an opening in metarhodopsin II between transmembrane helix
5 (TM5) and TM6, in TM3 near E122, and in the “retinal plug”
formed by extracellular loop 2 (EL2). The majority of these sites
are more proximal to the β-ionone ring of retinal than the Schiff
base, indicating their influence on retinal release is more likely
due to steric effects during retinal dissociation, rather than alterations
to Schiff base stability. An Arrhenius plot of zebrafish rhodopsin
was consistent with this model, inferring that the activation energy
for Schiff base hydrolysis is similar to that of bovine rhodopsin.
Functional variation at key sites identified in this study is consistent
with the idea that retinal release might be an adaptive property of
rhodopsin in vertebrates. Our study is one of the few investigating
a nonmammalian rhodopsin, which will help establish a better understanding
of the molecular mechanisms contributing to vision in cold-blooded
vertebrates.
%I ACS Publications