This project will develop the basis for structure analysis of PDGF-AA in Phase I, leading to its 3D structure determination in PHase II. The ultimate goal of this grant is to use the 3D structure of PDGF to develop drugs that inhibit the actions of PDGF-AA in atherosclerosis. This approach will take advantage of the most contemporary techniques in biotechnology and structure analysis. Milligram quantities of PDGF AA-homodimer will be produced by recombinant DNA methodology; the cloned protein will be expressed in E. coli, followed by its renaturation and final purification. Biologically active PDGF-AA will then be used to grow homodimer crystals suitable for single crystal x- ray diffraction studies. NMR spectroscopy will be pursued concurrently on the protein in solution, in order to establish the basis for 2D-NMR analysis of its structure. Crystals suitable for x-ray diffraction studies to high resolution and/or NMR spectral data suitable for planning a detailed 3D structure analysis would form the basis of a Phase II strategy. The feasibility of using computational algorithms to expedite modeling will also be assessed. A detailed 3D structure of PDGF AA-homodimer would be determine in the Phase II program, leading to a molecular basis for rational design of drugs that block PDGF action in the development of atherosclerotic plaque.
