| Abstract
| - We demonstrate here that a nitrile-derivatized phenylalanine residue, p-cyanophenylalanine (PheCN), andtryptophan (Trp) constitute a novel donor−acceptor pair for fluorescence resonance energy transfer (FRET).The Förster distance of this FRET pair was determined to be ∼16 Å and hence is well suited for determiningrelatively 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-derivatizedmutants of the MPx peptide, each containing a PheCN residue that replaces different positions along the peptidesequence (i.e., from position 5 to 11) and serves as a resonance energy donor to the native Trp residue atposition 3, were studied spectroscopically. The FRET efficiencies obtained from these peptides allowed us togain 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 ∼4.2 Å, whereas in 7 M urea the radius of gyration increases to ∼6.5 Å, indicatingthat the peptide conformations become more extended under this condition. However, we found that temperaturehad only a negligible effect on the size of the MPx peptide, underlining the difference between the thermallyand chemically denatured states of polypeptides. The application of the Gaussian chain or the wormlike chainmodel allowed us to further obtain the probability distribution function of the separation distance betweenany 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 Å in water and 4.28 Å in 7 M urea.
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