Intramolecular quenching of tryptophan fluorescence by the peptide bond in cyclic hexapeptides

Intramolecular quenching of tryptophan fluorescence by protein functional groups was studied in a series of rigid cyclic hexapeptides containing a single tryptophan. The solution structure of the canonical peptide C[D-PpYTFWF] (pY, phosphotyrosine) was determined in aqueous solution by 1D- and 2D-1H NMR techniques, The peptide backbone has a single predominant conformation. The tryptophan side chain has three chi(1) rotamers: a major chi(1) = -60degrees rotamer with a population of 0.67, and two minor rotamers of equal population. The peptides have three fluorescence lifetimes of about 3.8, 1.8, and 0.3 ns with relative amplitudes that agree with the X, rotamer populations determined by NMR. The major 3.8-ns lifetime component is assigned to the chi(1) = -60degrees rotamer. The multiple fluorescence lifetimes are attributed to differences among rotamers in the rate of excited-state electron transfer to pepticle bonds. Electron-transfer rates were calculated for the six preferred side chain rotamers using Marcus theory. A simple model with reasonable assumptions gives excellent agreement between observed and calculated lifetimes for the 3.8 and 1.8-ns lifetimes and assigns the 1.8-ns lifetime component to the chi(1) = 180degrees rotamer. Substitution of phenylalanine by lysine on either side of tryptophan has no effect on fluorescence quantum yield or lifetime, indicating that intramolecular excited-state proton transfer catalyzed by the c-ammonium does not occur in these peptides.