Peptide sequence and conformation strongly influence tryptophan fluorescence.
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This article probes the denatured state ensemble of ribonuclease Sa (RNase Sa) using fluorescence. To interpret the results obtained with RNase Sa, it is essential that we gain a better understanding of the fluorescence properties of tryptophan (Trp) in peptides. We describe studies of N-acetyl-L-tryptophanamide (NATA), a tripeptide: AWA, and six pentapeptides: AAWAA, WVSGT, GYWHE, HEWTV, EAWQE, and DYWTG. The latter five peptides have the same sequence as those surrounding the Trp residues studied in RNase Sa. The fluorescence emission spectra, the fluorescence lifetimes, and the fluorescence quenching by acrylamide and iodide were measured in concentrated solutions of urea and guanidine hydrochloride. Excited-state electron transfer from the indole ring of Trp to the carbonyl groups of peptide bonds is thought to be the most important mechanism for intramolecular quenching of Trp fluorescence. We find the maximum fluorescence intensities vary from 49,000 for NATA with two carbonyls, to 24,400 for AWA with four carbonyls, to 28,500 for AAWAA with six carbonyls. This suggests that the four carbonyls of AWA are better able to quench Trp fluorescence than the six carbonyls of AAWAA, and this must reflect a difference in the conformations of the peptides. For the pentapeptides, EAWQE has a fluorescence intensity that is more than 50% greater than DYWTG, showing that the amino acid sequence influences the fluorescence intensity either directly through side-chain quenching and/or indirectly through an influence on the conformational ensemble of the peptides. Our results show that peptides are generally better models for the Trp residues in proteins than NATA. Finally, our results emphasize that we have much to learn about Trp fluorescence even in simple compounds.
author list (cited authors)
Alston, R. W., Lasagna, M., Grimsley, G. R., Scholtz, J. M., Reinhart, G. D., & Pace, C. N
complete list of authors
Alston, Roy W||Lasagna, Mauricio||Grimsley, Gerald R||Scholtz, J Martin||Reinhart, Gregory D||Pace, C Nick