Steven O. Smith
Professor and Director of Structural Biology
Department of Biochemistry and Cell Biology
Stony Brook University
312 Life Sciences Bldg.
Stony Brook, NY 11794-5215
1988 Searle Scholar
Membrane proteins serve an array of functions in cell membranes ranging from receptors involved in signal transduction to ion channels. The overall goal of our research is to understand the mechanisms of membrane proteins in chemical terms. Our current research on signal transduction mechanisms mediated by protein conformational changes involves the visual pigment rhodopsin, the G-protein coupled receptor responsible for vision at low light intensities. Absorption of light by the retinal chromophore of rhodopsin leads to a conformational change in the protein that allows binding of the G-protein transducin. Rhodopsin may serve as a model for the larger family of ligand-activated G-protein coupled receptors since the sequences and overall protein architecture are conserved. We have recently been able to position the retinal chromophore in the interior of the rhodopsin protein using NMR distance constraints. Based on a structural model of rhodopsin developed from these studies in combination with biochemical and molecular biological approaches, we have been able to establish the key residues that are in contact with the retinal and responsible for protein activation.
Our research on the mechanism of signal transduction mediated by receptor oligomerization has focused on the transmembrane and juxtamembrane domains of several membrane proteins involved in signaling. In these systems, the 'signal' is often transduced by ligand-induced dimerization of receptor ectodomains leading to tyrosine kinase activation of the intracellular domains. Current projects are on the structure of the transmembrane domain of the neu/erbB-2 receptor protein and the structure of the E5 protein and its interactions with the PDGF receptor. In these proteins, transmembrane interactions are known to be involved in the signal transduction process. In the case of the neu receptor, we have shown that a valine to glutamic acid substitution leads to constitutive activation of the receptor through strong hydrogen bonding interactions of the glutamic acid side chain carboxyl group.
Finally, a second important function of membrane proteins involves ion transport across cell membranes. Structure-function studies are in progress on phospholamban, a 52-reside membrane protein found in cardiac sarcoplasmic reticulum. The protein forms a pentameric complex in membranes and functions as a calcium-selective ion channel. The small size of the protein makes it tractable for detailed structural studies. Phospholamban shares many features with the much larger mammalian ion channels which may allow us to understand how these proteins are regulated in terms of ion specificity and gating. We have recently proposed a low resolution structure for the phospholamban pentameric complex that we are now solving to high resolution using NMR methods.
The structural tools used most often in our group are polized FTIR and magic angle spinning NMR. These methods provide a probe of the local secondary structure and the three dimensional packing of the transmembrane portions of membrane proteins. Magic angle spinning methods allow us to measure internuclear distances between 13C labels in a protein out to a distance of about 6.5 angstroms with resolution on the order 0.2 angstroms. Also, we have recently developed an IR method based on cysteine sulfhydryl exchange that allows us to determine the rotational orientation of transmembrane helices in a membrane protein or protein oligomer. Proposed mechanisms based on structures derived using these methods can subsequently be tested using biochemical and molecular biological approaches.
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