2020, Scheerer, David, Chi, Heng, McElheny, Dan, Keiderling, Timothy A., Hauser, Karin

Site-specific isotopic labeling of molecules is a widely used approach in IR-spectroscopy to resolve local contributions to vibrational modes. The induced frequency shift of the corresponding IR band depends on the substituted masses, but also on hydrogen bonding and on vibrational coupling. The impact of these different factors was analyzed with a designed three-stranded β-sheet peptide and by use of selected 13C isotope substitutions at multiple positions in the peptide backbone. Single strand labels give rise to isotopically shifted bands at different frequencies depending on the specific sites, demonstrating sensitivity to the local environment. Cross-strand double and triple labeled peptides exhibited two resolved bands, which could be uniquely assigned to specific residues, whose equilibrium IR indicated only weak local-mode coupling. Temperature-jump IR-laser spectroscopy was applied to monitor structural dynamics and revealed an impressive enhancement of the isotope sensitivity to both local positions and coupling between them as compared to equilibrium FTIR. Site-specific relaxation rates were altered upon introduction of additional cross-strand isotopes. Likewise, the rates for the global β-sheet dynamics were affected in a manner dependent on the distinct relaxation behavior of the labeled oscillator. The study demonstrates that isotope labels do not just provide local structural probes, but they rather sense the dynamic complexity of the molecular environment.

2008, Huang, Rong, Krejtschi, Carsten, Hauser, Karin, Keiderling, Timothy A.

Beta-hairpins may be the smallest folding units in a protein, and two antiparallel beta-strands connected by a turn make the simplest model system for analysis of the interactions and dynamics of betasheets. We have studied site-specific conformational dynamics by use of equilibrium and temperature-jump kinetic IR-spectroscopy with site-specific enhancement via isotopic labelling of the amide with 13C=O in isotopically labeled variants of a modification of Cochran’s 12-residue tryptophan zipper peptide, TrpZip2. Equilibrium measurements reflect decreased stability of the hairpin crossstrand H-bonds at the turn and the termini. Spectral analysis of single and doubly labeled species is used to determine specific coupling levels. 13C=O groups introduced at different amide positions lead to distinguishable cross-strand coupling of the labelled residues which is lost on unfolding. These labels have distinct frequency patterns and different thermal behaviors depending on their position in the hairpin and reflect the local structural variation along the strands. Relaxation kinetics upon laser-induced T-jumps of ~10 C have time constants of a few microsec that decrease with ncrease of the initial temperature of the peptide before the temperature jump. Analysis of the data supports a multistate folding process, consistent with the hydrophobic collapse hypothesis for hairpin folding, but it is not possible to clearly define a folding and unfolding rate.