Site-specific folding dynamics of isotopically labeled peptides studied by time-resolved infrared-spectroscopy

Abstract

Peptides with well-defined secondary structure are ideal model systems for study of protein folding dynamics for specific, unique structures. IR techniques provide the necessary time resolution as well as have structural sensitivity, which arises from coupling of sequential residues, normally evidenced as a splitting or frequency shift of the amide centered transitions. The amide I region, mainly the C¼O stretching vibrations of the polypeptide backbone, is the prime target band for secondary structure. Isotopic labeling of individual amide 13C¼O groups can induce site-specific frequency shifts and provide insight into local structure. A nanosecond laser is used to excite the solvent and induce a fast temperature jump (~10 C), and relaxation dynamics are probed with a diode laser tuned to selected, structurally sensitive wavenumbers across the amide I absorption. Site-specific dynamics have been monitored for the thermal unfolding of an isotopically labeled beta-hairpin peptide, a 12-mer tryptophan zipper peptide, which has a hydrophobic core formed by four Trp residues, by use of cross-strand coupled 13C¼O labeled variants [1]. Data for single labeled peptides provided a control. Mutants of this sequence with just two Trp residues were introduced to destabilize the hairpin selectively near the termini or near the turn. Differences in kinetic behavior have been found for the loss of beta-strand and the gain of disordered structure. The isotope-edited kinetics vary with labeling position along the hairpin backbone and the mutations show consistent patterns depending on position. Our data supports a multistate folding mechanism for this hairpin structure. Similarly obtained data for other model peptides provide useful basis for interpretation of the observations.