Steady state and time resolved fluorescence spectroscopy techniques have been used to study mutants of bacteriorhodopsin and rhodopsin proteins. We have used site-directed mutagenesis to introduce two unique, derivatizable cysteines into the cytoplasmic loops of rhodopsin. We then attached the fluorescent reporter group pyrene maleimide to these cysteines, which might form excimer. The excimer spectrum is only observed with the two pyrenes and the cysteines they are attached to are close together, i.e. ~ 5 Angstroms. We have observed excimer fluorescence from a pyrene labeled control mutant, V227C/K231C. With this approach we are attempting to map out the proximity of residues in neighboring helices and within the cytoplasmic loops of rhodopsin. In the second project we use photocross-linking reagents. The photoreactive trifluorodiazarine in DTDA was then photoactivated using the Nd:YAG laser (lambda=355 nm, 7 nsec/pulse, 58 mJ/pulse). The goal was to detect where conformational changes occur in the loops and where the G-protein Transducin binds to the loops. In one series of experiments, we showed that the DTDA photocrosslinking agent attached at residue 240 in the loops was able to photocrosslink to transducin, indicating that there is a close contact of the alpha subunit of transducin with this region of rhodopsin [Resek et al., 1994].

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Shih, Wei-Chuan; Bechtel, Kate L; Rebec, Mihailo V (2015) Noninvasive glucose sensing by transcutaneous Raman spectroscopy. J Biomed Opt 20:051036
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