USING 7-AZATRYPTOPHAN TO PROBE SMALL MOLECULE-PROTEIN INTERACTIONS ONTHE PICOSECOND TIME-SCALE - THE COMPLEX OF AVIDIN AND BIOTINYLATED 7-AZATRYPTOPHAN

The utility of 7-azatryptophan as an alternative to tryptophan for opt ically probing protein structure and dynamics is demonstrated by inves tigating the complex of egg-white avidin and biotinylated 7-azatryptop han. We report the synthesis of biotinylated 7-azatryptophan and optic al measurements of its complex with avidin. Although there are four bi otin binding sites, the emission from the 7-azatryptophan tagged to bi otin decays by a single exponential, whereas the tryptophyl emission f rom avidin requires two exponentials in order to be adequately fit. Fl uorescence depolarization measurements of the complex probed by emissi on from 7-azatryptophan reveal both rapid (similar to 80 ps) and much longer-lived decay. The former component is attributable to the local motion of the probe with respect to the protein; the latter component represents overall protein tumbling. In addition, energy transfer from tryptophan to 7-azatryptophan and a blue-shift in the spectrum of bio tinylated 7-azatryptophan are observed upon formation of the complex. Modified strategies of effecting optical selectivity are also discusse d.