A new time-resolved fluorescence spectrophotometer was assembled, tested and modified. It was converted to a """"""""T"""""""" format for simultaneous acquisition of polarization and decay data. While many applications planned for this instrument require interfacing and computing links still under construction, it has already proved valuable in the analysis of protein fluorescence. It produces data that, when combined with """"""""global analysis"""""""" programs (see references), provides closer decay resolutions than previous instruments. Previously undisclosed components were found in proteins such as HLADH and BSA. The enzyme glutamine synthetase (GS) from the laboratory of Ann Ginsburg was studied. It was found to undergo a specific conformational response to inactivation in a ternary complex with cofactor ADP. Very closely related analogs of ADP were, in constrast, unable to effect the same tryptophyl site perturbation. Recently, the instrument was used to measure a sixfold slowing of Brownian rotations tha accompanies the binding of mellitin to calmodulin. The polarization decay capabilities of the instrument were also used to verify the rigid binding of a new bifunctional fluorescent label within IgG molecules. More recently, the subnanosecond resolution of the instrument was employed to characterize the exclusively excited-state protonation of serotonin. A theoretical framework was devised that should apply to other trp derivatives. In addition, a variety of smaller projects were begun or participated in: - autocorrelation methods for decay instruments. - excited state deprotonation mechanism of popular FACS pH indicator """"""""DCH"""""""". - Shape (axial ratio) of HLADH from excitation tuning (with globals) - mechanism of the self quenching of dyes inside liposomes.
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