Baenziger, J.,Méthot, Nathalie.2009-03-232009-03-2319991999Source: Dissertation Abstracts International, Volume: 61-01, Section: B, page: 0249.9780612465336http://hdl.handle.net/10393/8801http://dx.doi.org/10.20381/ruor-7488The goal of the research was to investigate the secondary structure and orientation of the channel forming transmembrane segments in the nicotinic acetylcholine receptor (nAChR) and their change in orientation upon desensitization. The long term objective is to further characterize the desensitized state of the nAChR as a basis of better understanding how the modulation of synaptic transmission is involved in brain function. FTIR spectroscopy was first used to probe the secondary structure of the nAChR and the changes in structure induced upon channel inactivation. Both qualitative and quantitative analysis of the secondary structure-sensitive amide I band of FTIR spectra of the nAChR support a mixed alpha/beta type protein with a predominance of alpha-helices. Stabilization of the nAChR in channel inactive conformations by either prolonged exposure to the agonist carbamylcholine, to the non-competitive antagonist tetracaine or upon reconstitution into membranes lacking anionic phospholipids and cholesterol revealed no changes in the amide I band, hydrogen/deuterium exchange kinetics or exchange difference spectra attributed to a change in protein conformation. Several approaches were directed toward the elucidation of the secondary structure of the transmembrane segments in the nAChR. Hydrogen/deuterium exchange experiments revealed the presence of an exchange resistant core of hydrogen peptides, corresponding to roughly 25% of the protein, mainly alpha-helical, and likely located in the solvent inaccessible transmembrane domain. 1H/2H exchange experiments at alkaline pH and upon reconstitution of the nAChR in egg phosphatidylcholine membranes to increase the rate and extent of exchange were performed and support a high proportion of alpha-helical peptides in the exchange resistant core of the nAChR. The amide I band of FTIR spectra of the transmembrane domain of the nAChR isolated by pronase under membrane disruption conditions (less than 3.7 kDa in size) revealed a very high content of alpha-helices. Similarly, we analyzed the secondary structure of individual transmembrane segments from the nAChR isolated by Dr. Michael Blanton at Texas Tech University and observed a high alpha-helical content. Taken together, these results support an alpha-helical transmembrane domain in the nAChR. Polarized FTIR spectroscopy revealed, for the first time, an average calculated tilt angle of roughly 41° of the transmembrane segments in proteolyzed nAChR and no detectable net change in orientation was observed upon carbamylcholine induced desensitization.199 p.Biology, Neuroscience.Thesis