STRUCTURE/INTERACTIONS OF ACTINS & ACTIN-BINDING PROTEIN

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The long range goal of this project is to determine at atomic resolution the three-dimensional structures of selected proteins of the actin system with the expectation that this information will provide the key to understanding how actin-based contractile and cytoskeletal systems are assembled ad how they function. To reach this goal we propose the following objectives for the next five years. Collect additional x-ray data and refine the structure of Acanthamoeba profilin-I to a resolution of 2.0 A or better. Analyze possible structural and sequential similarities between profilin and other actin-binding proteins and between profilin and lysozyme, using programs for comparing DNA or protein sequences or for matching up structures. Determine the structure of existing crystals of Acanthamoeba profilin-II at a resolution of 2.4A or better using native x-ray diffraction data and molecular replacement ot calculate initial phases. Determine the binding sites(s) on profilin-I for polyproline by x-ray crystallography of co-crystals. Determine the structure of human platelet profilin at atomic resolution by either molecular replacement or heavy atom methods. When available this information will be used to assist with determination of the structure of the vertebrate actin- profilin compelx. Small crystals of human platelet profilin are already available. Determine the structure of Acanthamoeba actophorin, a 15,000 Dalton actin filament severing protein, by x- ray crystallography using multiple heavy atom isomorphous replacements to calculate initial phases. Small crystals of actophorin are already available. Continue our encouraging initial efforts to prepare crystals, suitable for x-ray diffraction, of other proteins in the cytoskeleton including Acanthamoeba actin, covalently crosslinked complexes of Acanthamoeba actin with profilin-I or profilin-II, and a 70,000 Dalton fragment of the head of Acanthamoeba myosin-II. We will also attempt to crystalize rabbit cardiac muscle myosin subfragment-1 and the essential and regulatory light chains of smooth muscle myosin. We will attempt to crystalize oather proteins in the actin system as they become available. Make 3-dimensional reconstructions from electron micrographs of single actin filaments prepared in various ways in a effort to arrive at a consensus model for the filaments and to determine whether the polymerization conditions influence the structure of the polymer in some subtle way. Provide Acanthamoeba profilin and detailed structural information to collaborators who will use two-dimensional NMR to investigate the solution structure of the molecule and its repsonse to binding of polyproline and actin peptides for comparison with our x-ray crystallographic structure.