Conformational effects on tryptophan fluorescence in cyclic hexapeptides

Abstract

The peptide bond quenches tryptophan fluorescence by excited-state electron transfer, which probably accounts for most of the variation in fluorescence intensity of peptides and proteins. A series of seven peptides was designed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structure and side-chain χ1 rotamer populations of the peptides were determined by one-dimensional and two-dimensional 1H-NMR. All peptides have a single backbone conformation. The φ, ψ-angles and χ1 rotamer populations of tryptophan vary with position in the sequence. The peptides have fluorescence emission maxima of 350-355 nm, quantum yields of 0.04-0.24, and triple exponential fluorescence decays with lifetimes of 4.4-6.6, 1.4-3.2, and 0.2-1.0 ns at 5°C. Lifetimes were correlated with ground-state conformers in six peptides by assigning the major lifetime component to the major NMR-determined χ1 rotamer. In five peptides the χ1 = -60° rotamer of tryptophan has lifetimes of 2.7-5.5 ns, depending on local backbone conformation. In one peptide the χ1 = 180° rotamer has a 0.5-ns lifetime. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence.