Conformational Distribution of a 14-Residue Peptide in Solution:  A Fluorescence Resonance Energy Transfer Study

Abstract

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.

Equilibrium unfolding of the poly(glutamic acid)20 helix

“…Studies on the unfolded states of polypeptides, as well as loop regions in folded proteins, have indicated that unfolded conformations are flexible and can be reasonably approximately as a random coil, with persistence lengths and effective bond lengths 3.81. 34,66 However, the ABZ-E 20 -YNO 2 persistence length of >5.2 Å in pH 6 and 7.2 Å in pH 9 adds to increasing evidence that many unfolded proteins and peptides systems are more extended than theoretical random coils (l p > 3.8 Å ). 2,7,8,13,58 A recent study found that the persistence length of a number of regions of the natively disordered protein a-synuclein ranged between 5.5-6.5 Å .…”

“…11,13,14,[30][31][32][33] Many other studies have demonstrated that FRET is capable of measuring relatively small distance and distance distribution changes during conformational shifts in short peptide systems such as glycopeptide folding, glycosylation of peptide substrates, thermal stretching of unfolded peptides, zinc finger folding, hydrophobic peptide collapse, and a-helix denaturation. [16][17][18][19][20][21]34 However, to date, a detailed denaturation titration between a 100% a-helical peptide and a 0% helix random coil state has not been conducted using FRET to investigate the degree to which this transition is indeed a simple two-state transition or whether alternate conformations may exist. This is the objective of the present study.…”

Thermodynamics of protein folding using a modified Wako-Saitô-Muñoz-Eaton model

“…The GB1-m3 peptide was designed with a much better thermal stability than the parent GB1 peptide [70]. Tucker et al designed a new FRET pair on the GB1-m3 peptide by replacing the single Phe residue with a non-natural residue, Phe CN , where a cyano group is added at the para position of the Phe side chain [75]. The FRET pair is therefore formed between the Phe CN (donor) and the Trp (acceptor).…”

The alanine-rich XAO peptide adopts a heterogeneous population, including turn-like and polyproline II conformations

“…Recently, Jun et al (34) used electron spin resonance distance measurements to probe the distance of two spin labels attached to an alanine-based peptide in the folded and (thermally) unfolded states and found the respective value for the latter to be too short for a pure PPII state of the peptide. Analogously, Tucker et al (35) used an intrinsic FRET pair to deduce that the supposedly disordered mastoparan X peptide actually exists in aqueous solution as an ensemble of compact conformations.…”