THE MECHANISMS OF IMPORTANT CELLULAR PROCESSES SUCH AS TRANSMEMBRANE SIGNALING ARE NOT UNDERSTOOD AT THE MOLECULAR LEVEL BECAUSE OF THE DIFFICULTY IN OBTAINING STRUCTURAL INFORMATION ON MEMBRANE PROTEINS WITH THE CLASSIC APPROACHES OF X-RAY CRYSTALLOGRAPHY AND SOLUTION NMR. HOWEVER, SEVERAL RECENTLY DEVELOPED SOLID-STATE NMR TECHNIQUES FOR MEASURING SELECTED INTERNUCLEAR DISTANCES CAN BE APPLIED TO STUDY THE LOCAL STRUCTURE OF ACTIVE SITES IN MEMBRANE PROTEINS. THE MEMBRANE-BOUND RECEPTORS OF BACTERIAL CHEMOTAXIS ARE AN IDEAL SYSTEM IN WHICH TO PROBE THE ROLE OF CONFORMATIONAL CHANGES IN THE MECHANISM OF TRANSMEMBRANE SIGNALING. THE PROPOSED RESEARCH COMBINES EMERGING SOLID-STATE NMR TECHNIQUES WITH BIOCHEMISTRY AND MOLECULAR BIOLOGY TO CHARACTERIZE LOCAL STRUCTURE. THE ROTATIONAL RESONANCE NMR TECHNIQUE WILL BE EMPLOYED TO MEASURE SPECIFIC 13C/13C DISTANCES IN DIFFERENT SIGNALING STATES OF THE CHEMOTAXIS RECEPTORS. THE SELECTIVITY OF THE PROPOSED DISTANCE MEASUREMENTS IS OBTAINED BY MEASURING DISTANCES INVOLVING A UNIQUE ISOTOPIC LABEL, EITHER IN THE RECEPTOR LIGAND, OR IN A UNIQUE AMINO ACID, CYSTEINE, INTRODUCED ANYWHERE IN THE PROTEIN BY SITE-DIRECTED MUTAGENESIS. THE TECHNIQUE IS CURRENTLY LIMITED TO DISTANCES BETWEEN THE UNIQUE LABEL AND AN AMINO ACID THAT OCCURS RARELY IN THE PROTEIN SEQUENCE (BE15 TIMES) . OUR STUDIES ALSO AIM TO BYPASS THIS LIMITATION BY DEMONSTRATING THE USE OF SINGLE-SITE ANALYSIS AND OF DOUBLE CYS DISTANCE MEASUREMENTS. THE PROPOSED EXPERIMENTS WILL FOCUS ON (1) THE LIGAND BINDING SITE AND (2) PROPOSED CONTACTS BETWEEN THE TRANSMEMBRANE HELICES; SUBSEQUENT STUDIES WILL FOCUS ON THE LESS UNDERSTOOD CYTOPLASMIC DOMAIN OF THE RECEPTORS. THUS WE AIM TO PROVIDE A DETAILED PICTURE OF PROPOSED CONFORMATIONAL CHANGES AND YIELD DIRECT INSIGHT INTO THE MECHANISM OF TRANSMEMBRANE SIGNALING.
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