SUBPICOSECOND FLUORESCENCE DEPOLARIZATION STUDIES OF TRYPTOPHAN AND TRYPTOPHANYL RESIDUES OF PROTEINS

Fluorescence depolarization of the exposed tryptophanyl residue in the peptide melittin and the buried tryptophanyl residue in the protein azurin (from the bacteria Pseudomonas aeruginosa) was measured with subpicosecond resolution at various excitation and emission wavelengths. A short time component was found in both the fluorescence decay and fluorescence anisotropy decay for the tryptophanyl residue in melittin with time constants of 1.4 +/- 0.2 and 3.8 +/- 2 ps, respectively. The short time components originate from internal conversion between the lowest lying singlet excited states. The limiting value of the initial anisotropy of 0.40 +/- 0.02 is observed for the tryptophanyl residue in melittin when excited with 300-nm light. These results are similar to those observed for tryptophan in water. The fluorescence depolarization measured for the tryptophanyl residue in azurin did not exhibit a short time component, which is attributed to both singlet excited states being nearly degenerate and the rate of internal conversion beyond our temporal resolution. We propose a new model for the fluorescence depolarization observed in the first few picoseconds for tryptophan in water and for tryptophanyl residues in proteins, which takes into account conformational heterogeneity and the effect this has on population exchange between the two low-lying singlet excited states.