The objective of this project is to extend the applications of optical spectroscopy, especially circular dichroism (CD), in the study of macromolecular structure and function. CD spectroscopy has undergone a major renaissance during the past decade. CD is increasingly popular because it is highly sensitive to protein and nucleic acid conformations and to ligand binding; its demands in quantity of sample are modest; and it lends itself well to surveying a broad range of solution conditions and temperature. CD and NMR are the most widely used techniques for studying protein folding and conformational transitions in DNA and RNA. CD is also of great value in studies of protein-DNA interactions. Studies in two basic areas are proposed: (1) the intrinsic CD of proteins, considering both backbone and side-chain contributions; (2) extrinsic CD contributions of hemes in heme proteins. New parameters, based upon ab initio wave functions for the peptide group will be developed and incorporated in calculations of the CD of alpha-helix, beta-sheet and poly(Pro)II conformations, as well as proteins of known structure. Through-bond coupling and polarizable group models for high energy transitions will also be introduced in the calculations. The CD spectra of small peptides - blocked alpha-amino acids alpha-di- and tripeptides, and beta-hexa- and heptapeptides- will be calculated from MD trajectories. Improved ab initio- based parameters will be developed for side chains groups , especially disulfides. Molecular dynamic simulations will be coupled with the improved CD calculations for several small proteins. Aromatic side- chain contributions to the CD of several proteins will be investigated in detail using improved parameters. The equilibrium and kinetics of folding and unfolding of a parallel beta-helix protein, wild-type and mutants, will be investigated by CD and fluorescence. The contribution of heme nonplanarity to the visible and near-uv CD of heme proteins will be investigated. The relative importance of intrinsic heme-aromatic, and heme-peptide interactions will be assessed for a wide range of heme proteins of known structure. Heme-heme interactions will be calculated for multi-heme proteins. Heme isomerism in cytochrome b5 will be studied experimentally and by MD-CD calculations. Exciton coupling of porphyrins in bisporphyrin steroids will be studied by calculations of CD for structures obtained by a stochastic dynamics simulation. Models of multi-heme proteins obtained by protein design (maquettes) will be studied experimentally and theoretically.
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