Through the broad, long term objectives of this research application, we seek to understand the mechanisms by which biological macromolecules recognize their partners in the formation of macromolecular assemblies. Systematic investigations are proposed that focus on the major classes of recognition events in biological systems according to the following Aims. (1) Protein - Protein Recognition. Here we seek to define the role of specific surface interactions; electrostatic, hydrogen bonding, and hydrophobic free energies provided through surface complimentary, which define the specificity and affinity in the formation of complexes between the metalloproteins coordinating electron transfer events in the eukaryotic cytochrome P450 dependent oxygenases. (2) Membrane component recognition. Biological membranes, with their complex makeup of phospholipids, fatty acids, cholesterol and other critical cellular components, are more than a simple """"""""detergent"""""""" for embedded eukaryotic P450 protein components. The composition and physical properties of the bilayer are important for the very recognition events that are the subject of Aim (1). Multiple techniques are brought to bear toward the goal of understanding the contributions of membrane architectures to molecular recognition. (3) Protein - Small Molecule Recognition. In this Aim we seek to ascertain how the same fundamental forces of electrostatics, hydrogen bonding and the hand - glove fit of a substrate into the active site of an enzyme can give rise to the observed high degree control of regio- and stereo- specificity in cytochrome P450 catalysis. (4) Protein - Nucleic Acid Recognition. Again the same fundamental forces control recognition processes. In this Aim we build on previous success in defining roles of solvent water in mediating hydrogen bond recognition between protein and nucleic acid components. We expand the repertoire of systems investigated by examining the interesting class of """"""""indirect readout"""""""" macromolecular assemblies and use novel hydrostatic and osmotic pressure methods, in concert with crystallography and molecular dynamics simulations. Our proposed research program in this competitive renewal make concerted use of broad interdisciplinary tools and techniques in molecular biology and advanced biophysical methods which have proven to be ideal for understanding the fundamental mechanisms of metalloenzyme mechanisms.
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