PHOTOPHYSICS OF THE SINGLE TRYPTOPHAN RESIDUE IN FUSARIUM-SOLANI CUTINASE - EVIDENCE FOR THE OCCURRENCE OF CONFORMATIONAL SUBSTATES WITH UNUSUAL FLUORESCENCE BEHAVIOR

The single tryptophan residue, at position 69 in the amino acid sequen ce, was used as an intrinsic probe to obtain structural and dynamical information on the lipolytic enzyme Fusarium solani cutinase. In the e nzyme's native state the tryptophan fluorescence is highly quenched. T ime-resolved experiments reveal that the majority of the excited state species is characterized by an unusually fast decay time of approxima tely 40 ps, indicating the occurrence of a very efficient nonradiative relaxation process, possibly via the adjacent disulphide bond or via the peptide bonds of a nearby loop. A minority of the excited state sp ecies relaxes on a nanosecond time scale. Irradiation of the enzyme in the tryptophan absorption band causes an increase by an order of magn itude of the fluorescence quantum yield. This increase is ascribed to a photo-induced, subtle structural change of a minor subset of species whose fluorescence is not highly quenched. The structural change is a ccompanied by a tightening of the local environment of the tryptophan moiety, as indicated by results from time-resolved fluorescence anisot ropy which reveal a complete disappearance of the segmental flexibilit y of the tryptophan moiety.