MAPPING OF RHODOPSIN TRANSDUCIN INTERACTION SITES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Rhodopsin is an integral membrane protein that is present in rod cells of the retina. A light-induced conformational change in rhodopsin is the initiating event in the signal transduction pathway leading to light perception. One critical event in this signaling pathway is the binding of the protein transducin to light-activated rhodopsin. Given the critical role this protein-protein interaction, the Khorana group has directed studies towards understanding the precise nature of contact sites between these two proteins. Since the interaction between transducin and rhodopsin is short-lived, the studies incorporate photocrosslinking to capture this interaction as a covalent complex, thus allowing for further purification and analysis of the complex. Specifically, the presence of the crosslinker on transducin facilitates location of the site of interaction with relatively high precision. The binding reaction is carried out in vitro using purified recombinant rhodopsin and transducin. A series of rhodopsin molecules have been generated which contain only one cysteine residue each. This cysteine provides a sulfhydryl group that reacts with one end of the bifunctional photoactivatable crosslinker NETDB. The opposite end of NETDB contains a diazirene group that can react with transducin following activation by UV light. Subsequent to crosslinking and removal of unbound transducin, crosslinked transducin is removed from rhodopsin by reduction with dithiothreitol. Reaction of Texas Red maleimide with the free sulfhydryl group of the bound crosslinker provides a sensitive means of detecting crosslinked peptides by reversed-phase chromatography after trypsin digestion of transducin. This analysis is performed for each independent rhodopsin cysteine variant. Two approaches to localize the crosslinked sites by mass spectrometry are being pursued. One approach utilizes off-line chromatography to initially isolate crosslinked peptides using the Texas Red chromophore. Isolated fractions are first analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) to identify peptides that have a mass increase consistent with the presence of the crosslinker. Any identified crosslinked peptides are then further analyzed by tandem mass spectrometry (MSMS) in order to determine the specific amino acid residue that is covalently bonded to the crosslinker. The second approach utilizes on-line chromatography tandem MS (LC-MSMS) to localize the crosslinked amino acid in an automated process. The first approach has the advantage of allowing more time for analysis of each candidate peptide. However it is possible that the extra handling steps could lead to loss of low abundance samples. The LC-MSMS approach minimizes sample loss due to handling and concentrates samples into chromatographic peaks, thereby potentially increasing sensitivity. However, the MSMS analysis time for each peptide is limited in this approach. The particular method to be used is determined based on the quality of data produced for each sample. We have performed MS/MS analysis on seven transducin digests isolated from crosslinking reactions with rhodopsin cysteine variants. HPLC fractions of all the variants were analyzed by nanoESI MSMS. Electroelution from SDS-PAGE gel bands has also been investigated as a method to recover the crosslinked protein. The Khorana group is currently developing an alternative strategy to purify cross-linked peptides, making use of selective enrichment of cross-linked peptides prior to tandem MS analysis; this approach should increase the yield of peptides.