PROBING PEPTIDE CONFORMATION USING IR SPECTROSCOPY

Mount Holyoke College, South Hadley MA

Investigators

Abstract

Determining the mechanism by which a polypeptide chain folds into a stable three dimensional conformation ("the protein folding problem") is one of the most important challenges of biochemistry and biomedical science. Not only is the solution to this problem prerequisite for Dr. Decatur's understanding of the molecular basis of biological function, it also underlies several important diseases which are caused by protein misfolding. This project addresses the protein folding problem by developing a method of studying protein conformation and dynamics combining specific isotope labeling, infrared spectroscopy, and rapid mixing kinetics techniques. First the conformation of alanine-rich peptides will be studied by FTIR spectroscopy. These peptides are classic model systems for investigating the conformation and dynamics of alpha helices. In this project, the relationship between backbone solvation and helix stability will be investigated by infrared spectroscopy. Because both solvent-backbone interactions and secondary structure affect the amide I vibrations of peptides, IR is a very sensitive probe for dissecting the relationship between solvation and conformation. FTIR spectra will be measured on specifically-labeled peptides in order to obtain conformation as a function of residue position. Next, these peptides will be studied by stopped-flow infrared spectroscopy. IR detection of rapid mixing is less common for the study of biomolecules than circular dichrosim or fluorescence detection, but, when this method is applied to specifically isotope-labeled peptides and proteins, conformational changes can be resolved to the residue level. The data from these experiments, combined with the equilibrium FTIR and temperature jump measurements, will be used to characterize the nucleation and propagation steps of helix formation. Finally, this method will be expanded to the study of a full protein. Apomyoglobin is a well-studied model for folding in single domain globular proteins. Helices involved in the low pH folding intermediate of apomyoglobin will be specifically labeled with 13C using expressed protein ligation. This series of labeled proteins can then be studied by steady state and stopped-flow IR methods to obtain descriptions of the folding pathway.