The work proposed will continue the development of UV resonance Raman spectroscopy (UVRRS) as a new technique for the study of biomolecular structure and function. We will develop new instrumentation and methodologies to examine protein structure and function. This study will probe specific aromatic amino acids in hemoglobin to detail environmental changes which accompany heme ligand binding. Changes in geometry and structure of the heme and its ligands will also be examined in order to correlate heme alterations to globin quaternary and tertiary changes. These studies will be based on careful UVRR characterization of model systems which will include excitation profile measurements of aromatic amino acids, dipeptides, and small proteins. We will examine the dependence of the Raman intensities and frequencies upon environment, and use this information in hemoglobin studies. We will examine electronic communication between the heme molecular orbitals and those of imidazoles bound to the iron in model heme complexes. We will develop a new technique called saturation Raman spectroscopy to probe aromatic amino acid environment and distances between aromatic amino acid residues. The degree of optical saturation of aromatic amino acid residues depends upon the proximity to other chromophores which can relax the residue either by Forster transfer or electron transfer. We will use visible wavelength RRS and FTIR to study iron-ligand bonding as well as ligand-globin interactions. We will attempt, for the first time, to use vibrational circular dichroism to study interactions between the distal heme residues and heme bound ligands such as azide.
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