The primary objective of this project is to explore the principles underlying the structural organization, stability and action of macromolecular assemblies. Towards this end detailed studies of the structure of several macromolecular assemblies not amenable to conventional crystallographic analysis will be undertaken. Structures to be studied include filamentous bacteriophages Pf1, M13 and fd, gap junctions, recA-DNA complexes and amyloid fibers. A molecular model for the coat protein of Pfl will be constructed using fiber diffraction data to 3.5 angstrom resolution. This model will be used as a basis for understanding the changes in helical symmetry caused by changes in temperature and binding of heavy atoms; for predicting the structure of the viral DNA; and determining the origin of flexibility in phage particles. A model for the coat proteins of fd and M13 will be constructed using fiber diffraction data to 7 angstrom resolution. Helical symmetry changes induced by changes in pH and amino acid sequence will be investigated using phage particles constructed by site-directed mutagenesis. A 350 angstrom long M13 microphage particle has been constructed that contains all the structural elements of a mature, native phage including five copies each of four minor structural proteins. Crystallization conditions for these particles will be screened in order to obtain crystals for electron microscope and X-ray crystallographic studies. Structural studies of the microphage particles will provide substantial information about the assembly and infectivity of filamentous bacteriophages as well as information about the initiation and termination of a helical assembly of identical proteins. Structural studies of gap junctions will focus on determining the mechanism for control of intercellular communication. In addition, the geometric constraints intrinsic to the organization of twisted fibers of macromolecules will be used to analyze the properties of several biological fiber systems including fibrin and collagen fibers. These constraints may provide a basis for understanding the control of diameter in these and other biological fibers.
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