Sriram Subramaniam

Current Institution
National Cancer Institute
Head, Biophysics Section
Laboratory of Cell Biology, NCI/CCR

Scholar: 1993

Awarded Institution
The Johns Hopkins University


Research Interests

Structural Studies of Signal Transduction by Seven-Helix Receptors

Cell membranes contain a variety of receptors that recognize and transmit signals from the exterior to the interior of the cell. We are trying to understand the chemistry of this process in "seven-helix" membrane receptors, a major family of signal transducing proteins. In the last year, we have continued our studies with bacteriorhodopsin and Drosophila rhodopsin as model seven helix receptors.

Isomerization of retinal by light is the first step in the transduction of light energy by proteins in the rhodopsin family. In bacteriorhodopsin, retinal isomerization initiates a series of protein conformational changes that pump a proton from the inside to the outside of the cell. From spectroscopic studies of a series of mutants carrying replacements at residues in contact with retinal, we have identified key interactions between retinal and protein that are important for energy coupling. To determine light-driven conformational changes, we have developed methods to trap and structurally characterize (by electron diffraction of two-dimensional crystals) intermediates that occur in the course of the photocycle. To trap specific intermediates in high concentration, we have taken advantage of mutations such as Asp 96>Gly, and Leu93>Ala which slow down specific steps of the photocycle without affecting other steps. Such experiments have begun to provide molecular "snapshots" of the protein as it is in the process of carrying out light transduction.

Light absorption in the eye initiates a series of reactions which involve the interaction of the visual pigment rhodopsin with different photoreceptor cell proteins such as transducin, arrestin, rhodopsin kinase and rhodopsin phosphatase. To understand structural aspects of the interactions of rhodopsin with key photoreceptor proteins, we are developing methods to trap and biochemically characterize intermediates generated by light absorption. Using the Drosophila eye as a model system, we have obtained evidence for three biochemically distinct intermediates in the insect visual cycle: a "thermally unstable" form of the intermediate metarhodopsin which either decays into retinal and opsin, or is converted into a "thermally stable" form of metarhodopsin generated by binding arrestin, and an "inactive" rhodopsin-like intermediate from which rhodopsin is generated in a process that involves arrestin release and rhodopsin dephosphorylation. These experiments are beginning to provide a biochemical foundation for the application of crystallographic methods to study the interaction of the various photoreceptor proteins with rhodopsin at different stages in the visual cycle.