| Time-resolved and steady-state fluorescence spectroscopy in conjunction with circular dichroism were employed to investigate secondary and tertiary structural features of proteins. Several single tryptophan (Trp) proteins with known crystal structures were examined both in solution and in the crystalline state.;The three neurotoxins, ;Time-resolved fluorescence measurements for erabutoxin b single crystals exhibited multiexponential decay kinetics (single Trp residue). The relative proportion of each decay time component was found to be dependent upon the angular orientation of the protein crystal, with respect to the vertical polarization of the incident laser beam. These experimental data provide evidence for Trp side chain rotamers in the protein crystal. Model functions, which simulated the orientational dependence of the decay component relative proportions, were consistent with this rationalization. These results are the first direct experimental evidence obtained by fluorescence, to confirm the "rotamer" model for the interpretation of Trp fluorescence decay in solution. The measurement of crotonase, apoazurin and holoazurin crystals which display a range of exponential decay kinetics, provide further evidence for Trp rotamers in single protein crystals. |
