This proposal uses IR spectral analysis specific probes in peptide models and in nitric oxide synthase to report on local structure features, with the goal of expanding or developing new methodology for extracting structural and dynamic information on proteins from their IR spectra. Because vibrational frequencies are sensitive to a wide range of chemical and electrostatic interactions, infrared (IR) spectroscopy is a powerful tool for detailed information about protein structure. The first component of this proposal seeks to use the amide I modes to get specific information about interactions between amino acid residues and their environments. Because transition dipole coupling results in the delocalization of the amide I vibrational wavefunctions of all amino acid residues coupled in a secondary structural element, the frequency of the amide I mode is sensitive to the conformation of the peptide backbone. Thus, measurements of amide I frequencies using IR spectroscopy have been used to determine protein secondary structure content, as well as to follow global dynamic motions of the polypeptide chain. This sensitivity to overall peptide conformation comes at a loss of the use of a particular amide I band of a specific residue within a helix as a probe of local environmental interactions. In this project, incorporation of specific isotopic labels will be introduced into model helical peptides; the amide I band from C13 labeled carbonyls will be clearly distinguishable from the amide I band which arises from the other (carbon-12) residues, and its exact frequency will reflect both its position within the helix and its interactions with surrounding residues. This technique will be used to quantitate the effect of helix environment on amide I frequency and to probe thermodynamics and kinetics of helix formation. The second component of this project involves using the IR spectroscopy to elicit structural information about the heme active site of nitric oxide synthase (NOS), which catalyzes the production of nitric oxide from L-arginine and diatomic oxygen. This enzyme is an essential player in the biological activity of nitric oxide in the brain, the endothelium and the immune system. However, the details of the mechanism or the structure of the heme active site of NOS are still largely unknown. It is proposed that by measuring the IR spectra of CO-bound NOS, in the presence and the absence of substrate and inhibitors, the chemical and electrostatic nature of the heme pocket will be elucidated.
| Silva, R A; Kubelka, J; Bour, P et al. (2000) Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution. Proc Natl Acad Sci U S A 97:8318-23 |
