Understanding the mechanisms of visual transduction, bleaching adaptation, and microbial phototaxis are all very basic questions intimately related to the process of life. An understanding, if possible, can only be achieved through the efforts of researchers in various areas covering almost the entire field of science. Our efforts will be focused in contributing to this very difficult field mainly through organic and bioorganic approaches. Thus it will necessarily be centered in interpreting and understanding the phenomena on an organic structural basis. As in the past, our major tool will be to use synthetic retinal analogs, the structures of which have been designed so that they can solve specific aspects of these complex phenomena. We have made over 90 different analogs, not counting the cis/trans isomers as separate compounds. Specifically we will investigate the following topics: (a) The tertiary structure of the visual pigment rhodopsin. This is a membrane protein, and despite many efforts it has not been obtained crystalline. We are putting a major effort in mapping out the tertiary structure by a recently developed efficient photoaffinity label, which will be placed in critical sites of the chromophore. (b)The mechanism of bleaching adaptation. This well-known phenomenon is far less understood than the transduction mechanism. We have found a good chemical handle which will for the first time give us a chemical handle to investigate the structural factors of the chromophore that lead to bleaching. (c) The isomerization of vitamin A to 11-cis-retinal, the visual pigment chromophore, is an important step performed by the recently discovered enzyme isomerase. By reacting various analogs with the isomerase, we plan to investigate the details of the isomerase mechanism. (d)The phototaxis of the halophilic bacterium H. halobium depends on two retinal proteins, sensory rhodopsin-I and 11, which cause attractive and repellent movements of the flagella. Again the incorporation of retinal analogs into these photoreceptors has provided us with powerful tools to study the basic mechanism leading to flagella movement. (d)The photoreceptor of the phototactic unicellular algae Chlamydomonas has been found to be most unique in that it is activated by retinal analogs in which the double bonds are fixed so that no isomerization can occur. Furthermore, phototaxis is even induced by simple molecules such as hexanal. We plan to isolate sufficient quantities of the photoreceptor protein, by genomic means or a combination of analog incorporation and molecular biology, so that in vitro biochemical and biophysical measurements can be performed to elucidate these most unexpected results.
Showing the most recent 10 out of 60 publications
