Three-pulse two-dimensional infrared spectroscopy (2D-IR) is a technique which is capable of resolving molecular structure (via angles between transition dipoles and vibrational coupling constants), dynamics (via variable time delays and homogeneous line shapes), and structural distributions on time scales down to femtoseconds. Cytochrome-c is a well-studied, fast-folding heme protein: in cyt-c folding studies, misfolded and ligand-exchanged structures have been implicated in a heterogeneous dynamic structural distribution associated with the folding events. It is proposed here to use 2D-IR to resolve the equilibrium and dynamic (folding) structural distributions of the peptide chain of cyt-c near the heme. Isotopic labeling of Met80 (a native heme ligand) and Phe82 residues with 13C/180 and 13C/160 carbonyls will be used to spectrally isolate the amide-I vibrations of this part of the chain which is close to the heine. 2D-IR of the labeled residues, which yields the distances and angles between them, will be used to determine the equilibrium structural distribution of native-sequence cyt-c and the perturbed equilibrium distributions of Phe82 mutants. After integration of a visible trigger pulse into the existing three-pulse infrared optical bench, denatured CO-heme cyt-c will be photolyzed to initiate folding to the native structure. 2D-IR of the labeled residues will be used to monitor the time-evolving structural distribution associated with cyt-c folding.
| Londergan, Casey H; Wang, Jianping; Axelsen, Paul H et al. (2006) Two-dimensional infrared spectroscopy displays signatures of structural ordering in peptide aggregates. Biophys J 90:4672-85 |
