Conformational distribution of a 14-residue peptide in solution: a fluorescence resonance energy transfer study.

We demonstrate here that a nitrile-derivatized phenylalanine residue, p-cyanophenylalanine (Phe(CN)), and tryptophan (Trp) constitute a novel donor-acceptor pair for fluorescence resonance energy transfer (FRET). The Förster distance of this FRET pair was determined to be approximately 16 A and hence is well suited for determining relatively short separation distances. To validate the applicability of this FRET pair in conformational studies, we studied the conformational heterogeneity of a 14-residue amphipathic peptide, Mastoparan X (MPx peptide), in water and 7 M urea solution as well as at different temperatures. Specifically, seven nitrile-derivatized mutants of the MPx peptide, each containing a Phe(CN) residue that replaces different positions along the peptide sequence (i.e., from position 5 to 11) and serves as a resonance energy donor to the native Trp residue at position 3, were studied spectroscopically. The FRET efficiencies obtained from these peptides allowed us to gain a global picture regarding the conformational distribution of the MPx peptide in different environments. Our results suggest that the MPx molecules exist in water as an ensemble of rather compact conformations, with a radius of gyration of approximately 4.2 A, whereas in 7 M urea the radius of gyration increases to approximately 6.5 A, indicating that the peptide conformations become more extended under this condition. However, we found that temperature had only a negligible effect on the size of the MPx peptide, underlining the difference between the thermally and chemically denatured states of polypeptides. The application of the Gaussian chain or the wormlike chain model allowed us to further obtain the probability distribution function of the separation distance between any two residues along the peptide sequence. We found that the effective bond length of the MPx peptide, obtained by using the Gaussian chain model, is 2.78 A in water and 4.28 A in 7 M urea.