Receptors are a diversified class of membrane proteins which in the rod cell includes rhodopsin, a member of the G-protein linked receptor family and the cyclic-GMP activated cation channel. A variety of other receptors such as the nicotinic acetylcholine receptor are also important in the functioning of the retina. While the roles of such receptors in cellular function are becoming increasingly well defined in no case has the detailed molecular mechanism of any receptor protein yet been elucidated. The general goal of this project is the development of FTIR based methods to obtain detailed molecular information about receptor function at the level of individual amino acid residues. In the past funding period important progress has been made in this direction which includes: a) Measurement of the FTIR difference spectra for all of the steps in the rhodopsin photocascade. b) The first FTIR measurements on a non-light triggered receptor. c) Demonstration that receptors can be studied under physiological conditions. d) Utilization of mutants for band assignments and structure-function studies. e) Development of advanced FTIR techniques for receptor studies. We propose to utilize these novel techniques to investigate a variety of retina based receptors as well as model polypeptides in the next funding period. In the case of rhodopsin, these studies will aim at determining structural changes in key residues involved in color regulation, G-protein activation, phosphorylation, protein insertion and folding. The interaction of rhodopsin with other components in the visual signal-transduction system such as the G-protein, rhodopsin kinase and arrestin will also be investigated. Possible common mechanisms which underlie receptor function will be studied by focusing on a variety of other suitable receptors and model system including sensory rhodopsin I, the nicotinic acetylcholine receptor, the colicin E1 channel fragment and synthetic polypeptides. Our recent work has revealed spectroscopic features which are shared by this diverse group of receptors. One possibility is that ligand binding, light absorption, voltage-dependent changes and pH alterations all result in the modulation of the ionization states of specific buried Asp/Glu residues that cause a change in the interaction and orientation of transmembrane alpha-helices. These studies will also provide a basis for studying at a molecular level the effects of anesthetics, drugs and other soluble molecules on receptor function. In general, the novel approaches being developed in this project should have an important impact on future understanding of a wide variety of signal-transduction systems in biology.
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