Cross-Strand Coupling and Site-Specific Unfolding Thermodynamics of a Trpzip -Hairpin Peptide Using 13C Isotopic Labeling and IR Spectroscopy

Conformational properties of a 12-residue tryptophan zipper (trpzip) -hairpin peptide (AWAWENGKWAWK-NH2, a modification of the original trpzip2 sequence) are analyzed under equilibrium conditions using ECD and IR spectra of a series of variants, singly and doubly C1-labeled with 13C on the amide CdO. The characteristic features of the 13CdO component of the amide I ′ IR band and their sensitivity to the local structure of the peptide are used to differentiate stabilities for parts of the hairpin structure. Doubly labeled peptide spectra indicate that the ends of the -strands are frayed and that the center part is more stable as would be expected from formation of a stable hydrophobic core consisting of four tryptophan residues, and supported by MD simulations. NMR analyses were used to determine a best fit solution structure that is in close agreement with that of trpzip2, except for a small variation in the turn geometry. The distinct vibrational coupling patterns of the labeled sites based on this structure are also well matched by ab initio DFT-level calculations of their IR spectral patterns. Thermal unfolding of the peptides as studied with CD spectra could be fit with an apparent two-state transition model. ECD senses only the tryptophan interactions (tertiary-like) and their overall environment, as shown by TD-DFT modeling of the Trp-Trp π-π * ECD. However, variation of the amide I IR spectra of 13C-isotopomers showed that the thermal unfolding process is not cooperative in terms of the peptide backbone (secondary structure), since the transition temperatures sensed for labele