Molecular Biology of Visual Pigments 
Jeremy Nathans, M.D., Ph.D. — Associate Investigator 
Dr. Nathans is also Associate Professor of Molecular Biology and Genetics and of Neuroscience at the Johns 
Hopkins University School of Medicine. His undergraduate work was in biology and chemistry at the 
Massachusetts Institute of Technology. He received a Ph.D. degree in biochemistry and later his M.D. 
degree at Stanford University. Before joining the staff at Johns Hopkins, Dr. Nathans spent a year as a 
postdoctoral fellow at Genentech. 
VISUAL pigments are the light-absorbing pro- 
teins that initiate phototransduction. Each 
consists of a chromophore, 1 l -cis retinal, joined 
to an integral membrane protein, opsin. The vi- 
sual pigments constitute one branch of a large 
family of cell surface receptors that transduce ex- 
ternal stimuli by activating G proteins. In the vi- 
sual system, the activated G protein stimulates a 
cGMP phosphodiesterase, and the resulting tran- 
sient decline in cGMP closes plasma membrane 
cation channels. 
Photon absorption by 1 \ -cis retinal causes it to 
isomerize from W-cis to all-fraws. The attached 
protein then undergoes a series of conforma- 
tional changes, leading ultimately to a form that 
interacts with the G protein. The changes under- 
lying visual pigment activation are likely to re- 
semble those that accompany hormone-receptor 
binding among the other members of this recep- 
tor family. 
Our laboratory is interested in three general 
areas related to the visual pigments: their struc- 
ture and function, the control of their expression, 
and their variation within the human population. 
Structure/Function Studies 
Several years ago we developed a system for 
producing large quantities of bovine rhodopsin 
by expression of cloned cDNA in tissue culture 
cells. As described below, we are using this sys- 
tem in conjunction with site-directed mutagene- 
sis to define those residues involved in protein 
conformational changes, in protein-chromophore 
interactions, and in protein stability. 
Because the retinal chromophore is sensitive to 
changes in its immediate environment, the 
various conformations of light-activated rhodop- 
sin each possess distinctive absorption spectra. 
By following the changes in spectral absorbance 
following photoactivation, one can determine 
the quantity and rates of formation and decay of 
each conformational intermediate. Charles Weitz, 
a postdoctoral fellow, has used this assay to map 
those amino acids that play an important role in 
rhodopsin's transition to the active conforma- 
tion, i.e., the conformation that interacts with the 
G protein. 
In one set of experiments, we sought to exam- 
ine the mechanism responsible for the pH depen- 
dence of this transition. Thirty years ago George 
Wald and his colleagues observed that low pH 
favors this transition and that the pH dependence 
was consistent with a mechanism in which pro- 
tonation of one or more histidines was tightly 
coupled to the transition. We therefore mutated 
one-by-one each of the six histidine residues to 
phenylalanine and monitored the ability of the 
mutant rhodopsin to form the active conforma- 
tion. All of the mutant proteins could bind to 11- 
cis retinal and form a light-sensitive pigment, but 
mutants in which histidine^'' was replaced 
either by phenylalanine or cysteine were unable 
to assume the active conformation. 
The simplest interpretation of this experiment 
is that the histidine'^" is the site at which proton- 
ation drives rhodopsin into its active conforma- 
tion. Dr. Weitz has pursued this observation by 
identifying mutants at other sites that have the 
converse property: they form the active confor- 
mation more efficiently than the normal protein. 
This second type of mutant therefore identifies 
amino acids that normally act to keep the protein 
in the inactive conformation. As most amino acids 
have little or no effect on this conformational 
transition, it should be possible to identify the 
handful of critical residues that control it. 
In the human retina, rhodopsin mediates vision 
in dim light, whereas a related set of visual pig- 
ments, the cone pigments, mediate bright light 
vision as well as color vision. The spectral proper- 
ties of the cone pigments have been of great inter- 
est to physiologists and psychologists and have 
been the object of investigation for over a cen- 
tury. Unfortunately, the instability and scarcity of 
these proteins make them difficult to study. Sev- 
eral years ago we isolated the genes that encode 
the human cone pigments. Using these reagents, 
Shannath Merbs, a graduate student, has recently 
succeeded in producing large quantities of the 
pigments and determining their precise absorp- 
tion spectra. 
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