Molecular Biology of Visual Pigments 
Jeremy Nathans, M.D., Ph.D. — Assistant Investigator 
Dr. Nathans is also Assistant 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. de- 
gree at Stanford University. Before joining the staff at Johns Hopkins, Dr. Nathans spent a year as a post- 
doctoral fellow at Genentech. 
VISUAL pigments are the light-absorbing pro- 
teins that initiate phototransduction. Each 
consists of a chromophore, 1 1-cis retinal, joined 
to an integral membrane protein. The visual pig- 
ments constitute one branch of a large family of 
cell surface receptors that transduce external 
stimuli by activating G proteins. In the visual sys- 
tem, the activated G protein stimulates a cGMP 
phosphodiesterase, and the resulting transient 
decline in cGMP closes plasma membrane cation 
channels. 
Photon absorption by 11 -cis retinal causes it to 
isomerize from 1 1-cis to all-trans. It then under- 
goes a series of conformational changes, leading 
ultimately to a form that interacts with the G pro- 
tein. The changes underlying visual pigment acti- 
vation are likely to resemble those that accom- 
pany hormone-receptor binding among the other 
members of this receptor family. 
Our laboratory is taking three general ap- 
proaches related to the visual pigments: investi- 
gations of their structure and function, the con- 
trol of their expression, and their variation within 
the human population. 
Structure/Function Studies 
Several years ago we succeeded in producing 
large quantities of bovine rhodopsin by expres- 
sion of cloned cDNA in tissue culture cells. We 
are using this system in conjunction with site- 
directed mutagenesis to define the chromophore 
binding pocket, the residues involved in protein 
conformational changes, and the surface and 
transmembrane topography of the protein. 
In one experiment, each of the 22 negatively 
charged amino acids (e.g., aspartate or gluta- 
mate) was changed to a neutral residue of identi- 
cal size (asparagine or glutamine) . We observed 
that only one of the mutant proteins differs dra- 
matically from the wild type in its behavior. 
When glutamate*'' is mutated to glutamine, the 
Schiffs base linking 1 1 -cis retinal to the protein 
loses its proton. The base is re-protonated upon 
addition of small anions (e.g., chloride) to the 
sample. 
Our interpretation of this experiment is that 
glutamate'*^ is normally the counterion that sta- 
bilizes the protonated Schiff's base, but in its ab- 
sence a small anion can serve as a surrogate. This 
observation is likely to be relevant to the mecha- 
nism of protein activation, because SchifFs base 
deprotonation is required for the protein to as- 
sume its active conformation. 
In a second experiment, Charles Weitz, a post- 
doctoral fellow, has examined a number of rho- 
dopsin mutants for their ability to assume the ac- 
tive conformation. Thus far, one mutant binds the 
chromophore and absorbs light normally but ap- 
pears to be locked in the inactive conformation. 
In a third set of experiments, Jimo Borjigin, a 
graduate student, is using insertional mutagene- 
sis to tag rhodopsin at predetermined sites. The 
tags consist of stretches of foreign amino acids 
that can bind to other proteins (e.g., antibodies). 
These modified rhodopsins should be useful for a 
variety of structural studies. 
The expression system is also being used by 
Shannath Merbs, a graduate student, to produce 
the human cone pigments — a related group of 
light receptors that mediate color vision. 
Control of Visual Pigment Gene Expression 
As an entree into the general question of retinal 
development, we are examining the control of 
the genes for visual pigment. Donald Zack, a 
postdoctoral fellow, in collaboration with Jean 
Bennett and John Gearhart at Johns Hopkins, has 
constructed a set of transgenic mice that carry 
sequences upstream of the bovine rhodopsin 
gene joined to a gene encoding (8-galactosidase, a 
convenient histochemical marker. Rhodopsin 
gene fragments as small as 230 base pairs direct 
expression of the reporter gene to the photore- 
ceptor cells. 
Curiously, a considerably larger fragment 
(2,100 bp) directs expression in a spatially non- 
uniform manner: a gradient of expression forms 
across the retina. This DNA fragment appears to 
be responding to a preexisting spatial gradient. 
This gradient may be involved in determining the 
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