properties and photochemistry, the mechanism for 
transmitting a photochemical signal from the core 
of the receptor to the surface, and the specific do- 
mains on the cytoplasmic surface that bind and acti- 
vate transducin. 
The basic approach of the laboratory is to recon- 
stitute heterologously expressed rhodopsin and 
transducin in defined in vitro systems. A multifac- 
eted approach using a variety of complementary bio- 
chemical, biophysical, and spectroscopic methods 
is employed to probe site-directed mutants. 
Spectroscopic Studies of Mutant Visual 
Pigments 
A central question in vision research involves the 
mechanism of visual pigment spectral tuning. 
Nearly all vertebrate visual pigments share a com- 
mon chromophore, 1 l-c/s- retinal. However, the ab- 
sorption maxima values of visual pigments range 
from near-ultraviolet to red. These spectral differ- 
ences must be represented in unique chromophore- 
opsin interactions. 
In humans the differences in absorption maxima 
of the cone pigments that underlie human red-green 
color vision must result from differences in the 
amino acid sequences of the respective opsin pro- 
teins. Fifteen amino acid substitutions distinguish 
the human green pigment (530 nm) from the red 
(560 nm). Three of these residues were suggested 
to produce this spectral difference through a ge- 
netic analysis of eight primate visual pigments. The 
amino acid at each of these three positions in the rod 
pigment rhodopsin (500 nm) matches that of the 
green pigment. 
The influence of these residues was tested experi- 
mentally in Dr. Sakmar's laboratory by substituting 
the amino acid residues of the red pigment into rho- 
dopsin. The spectral properties of a series of mutant 
pigments in which hydroxyl-bearing amino acids 
were introduced indicated that two of the three po- 
sitions in combination appear to account for about 
three-quarters of the absorption difference between 
the human green and red pigments. Thus tyrosine 
277 and threonine 285 appear to be involved pri- 
marily in red-green spectral tuning. However, other 
amino acid residues, including a serine at position 
180, are likely to contribute to lesser degrees. 
In rhodopsin, spectral tuning was shown not to be 
influenced by electrostatic interaction with carbox- 
ylates other than the Schifif base counterion. A neu- 
tral chromophore-binding pocket model in which 
dipole and hydrogen bonding interactions predomi- 
nate has been proposed for rhodopsin. A similar 
model is likely to apply to the green and red color 
pigments as well. A complete understanding of 
spectral tuning in the visual pigments will require 
detailed spectroscopic studies, including resonance 
Raman spectroscopy of mutant rhodopsins. 
Resonance Raman vibrational spectroscopy has 
been an important tool for studying the structures of 
the chromophores of visual pigments. In collabora- 
tion with Dr. Richard Mathies and Steven Lin, the 
laboratory developed a microprobe system to allow 
resonance Raman spectroscopy of microgram quan- 
tities of recombinant visual pigments. This tech- 
nique was employed to study the effects of substitu- 
tions of carboxylic acid groups in the third 
transmembrane helix of rhodopsin. The results con- 
firmed and supplemented the earlier observations 
concerning the role of glutamic acid 11 3 in rhodop- 
sin that acts to stabilize the positive charge of the 
protonated Schifif base chromophore linkage. Based 
on the structural information obtained in these stud- 
ies, a model of the chromophore-binding pocket of 
rhodopsin was proposed that will be used to direct 
further studies into the mechanism of wavelength 
regulation by visual pigments. 
Fourier-transform infrared (FT-IR) spectroscopy 
is a novel vibrational-difiference spectroscopic tech- 
nique that provides specific structural information 
about the molecular changes that are associated 
with the photoactivation of rhodopsin. In collabora- 
tion with the laboratory of Dr. Friedrich Siebert, FT- 
IR spectra have been obtained on a collection of 
rhodopsin mutants with substitutions of carboxylic 
acid groups. The FT-IR technique is extremely sen- 
sitive to protonations and deprotonations of mem- 
brane-embedded carboxylic acid groups of rhodop- 
sin that occur during its photobleaching pathway. 
Drs. Sakmar and Siebert hope to use a combination 
of site-directed mutagenesis and FT-IR spectroscopy 
to follow intramolecular proton transfers that 
accompany the formation of the active state of 
rhodopsin. 
Rhodopsin-Transducin Interactions 
Light-activated rhodopsin catalyzes guanine nu- 
cleotide exchange by transducin. It was previously 
shown by biochemical and flash photolysis studies 
of site-directed rhodopsin mutants that a highly con- 
served glutamic acid-arginine sequence in rhodop- 
sin was involved in transducin binding. The second 
and third cytoplasmic loops of rhodopsin were 
shown to be necessary to activate bound transducin. 
Dr. Sakmar's laboratory is interested in identifying 
specific domains of rhodopsin and transducin in- 
volved in these discrete binding and activation 
events. 
To facilitate the quantitative assay of rhodopsin- 
transducin interactions. Dr. Karim Fahmy has devel- 
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