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

Abstract

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.

A Concerted Tryptophanyl-adenylate-dependent Conformational Change inBacillus subtilisTryptophanyl-tRNA Synthetase Revealed by the Fluorescence of Trp92

“…It was observed that t 1 , but not the shorter components, seemed to increase with increasing TrpRS concentration. Similar increases were observed in t 1 by Willis et al (1994) upon formation of structure in a model peptide. This may be due to the formation of higher-order oligomers or aggregates of TrpRS and may be related to the observation of low-wavelength light scattering in UV spectra.…”

“…Considerable experimental evidence now exists to support this interpretation (Ross et al, 1992;. It is clear that the values of c i (or a i ) for each decay component vary with the formation of secondary structure, as has been demonstrated in helical peptide models (Willis et al, 1994) and in small b-sheet containing neurotoxins . From the magnitude of changes in the values of c i observed in the DAS of apo-and 4FW-AMP complexed TrpRS (Figure 7), we conclude that there is a variation in the conformation of the local secondary structure wherein Trp92 resides.…”

“…Formation of an a-helix in a peptide containing Trp was shown to have had a significant effect on the pre-exponential terms (c i or a i ) of the fluorescence decay with model peptides (Willis et al, 1994). The strongest feature observed upon helix formation was the increase in the value of c 1 of the long decay time component, and the decrease in value of c 2 of the intermediate decay time component.…”

Ultrafast Polarized Fluorescence Measurements on Monomeric and Self-Associated Melittin

“…This provides a tool to probe the secondary structure of proteins, as the fractional distribution of the rotamers depends on the main chain conformational constraints. 20,21 In this work we use time-resolved fluorescence depolarization to examine the self-association process of melittin. The use of a streak camera setup enables us to monitor the fluorescence decay of the complete emission spectrum of the Trp residue over a large time interval ranging from a few picoseconds to a few nanoseconds.…”

Trp42 rotamers report reduced flexibility when the inhibitor acetyl‐pepstatin is bound to HIV‐1 protease

“…The stereochemically favored rotamers of the x 1 conformation are the staggered forms, possible when x 1 ϭ 60, Ϫ60, and 1808; these rotamers are called the g ϩ , g Ϫ , and t rotamers, respectively. Similarly, the favored x 2 rotamers are the 908 and Ϫ908 forms~Szabo & Rayner, 1980;Willis et al, 1994!. …”