NMR, fluorescence, and computational studies of cyclic hexapeptides containing a single tryptophan

Tryptophan fluorescence is widely used to study the conformational change in proteins. However, this application was hindered by the complex photophysics, including fluorescence emission maxima, intensity, and lifetime. To address these complicated issues, a series of seven peptides was developed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structures of the peptides and chi 1 rotamer populations of the tryptophan side-chain were determined by 1D and 2D 1H-NMR. All peptides have a single backbone conformation. The &phis;, psi angles and chi1 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 4.5--6.7, 1.4--3.1, and 0.5--0.9 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 chi1 rotamer. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence.; The decay-associated emission spectra of peptides show a wide range of 339--359 nm emission maxima, suggesting that the tryptophan rotamers have different electrostatic environments. Hybrid quantum mechanics/molecular dynamics computations were performed to simulate the wavelength of maximum emission of the peptides where tryptophan side-chains were in six canonical rotameric conformations. The calculated emission maxima for peptides also show a wide range of 344--365 nm. The precision of calculated emission maxima was evaluated. The rotameric effects of tryptophan and other nearby polar side-chain on calculated emission maxima were revealed. Possible new correlations between lifetimes and ground-state conformers based on comparison of experimental emission maxima of DAS with the simulated wavelengths were discussed.