Probing alpha-helical secondary structure at a specific site in model peptides via restriction of tryptophan side-chain rotamer conformation.

The relationship between alpha-helical secondary structure and the fluorescence properties of an intrinsic tryptophan residue were investigated. A monomeric alpha-helix forming peptide and a dimeric coiled-coil forming peptide containing a central tryptophan residue were synthesized. The fluorescence parameters of the tryptophan residue were determined for these model systems at a range of fractional alpha-helical contents. The steady-state emission maximum was independent of the fractional alpha-helical content. A minimum of three exponential decay times was required to fully describe the time-resolved fluorescence data. Changes were observed in the decay times and more significantly, in their relative contributions that could be correlated with alpha-helix content. The results were also shown to be consistent with a model in which the decay times were independent of both alpha-helix content and emission wavelength. In this model the relative contributions of the decay time components were directly proportional to the alpha-helix content. Data were also analyzed according to a continuous distribution of exponential decay time model, employing global analysis techniques. The recovered distributions had "widths" that were both poorly defined and independent of peptide conformation. We propose that the three decay times are associated with the three ground-state chi 1 rotamers of the tryptophan residue and that the changes in the relative contributions of the decay times are the result of conformational constraints, imposed by the alpha-helical main-chain, on the chi 1 rotamer populations.