Our long-range goal is to understand the structure of the eukaryotic chromosome in general, the human chromosome in particular, and the regulation of differential gene expression at the molecular level. For the next five years, we want to focus on the structure-function relations of the basic 100 A and 300 A diameter chromosomal fibers, the nucleosome, and,its octameric histone core complex. This complex, composed of two of each of the four """"""""inner histones"""""""" H2A, H2B, H3, and H4, is responsible for the primary compaction of the DNA double helix in all eukaryotic organisms and must also be involved at least in some aspect of the regulation of chromosomal physiology. We will use a combined approach which includes X-ray crystallography, small-angle X-ray scattering (SAXS) of solution and paracrystalline chromatin systems, and hydrodynamic and thermodynamic measurements of the relevant histone and histone-DNA complexes. We already have collected single-crystal X-ray diffraction data sets from one native and two different isomorphous heavy atom derivatives of the histone octamer crystals, have calculated electron density maps, and started fitting alpha-helices into them. We also have collected preliminary SAXS data as well as made preliminary physicochemical measurements of the effects of solvent on the structure of the histone octamer. We propose to (a) complete the placement of amino acids in the electron density map and refine the modeled structure, (b) collect at a synchrotron the highest resolution data possible from the crystals at hand and new histone octamer crystals, (c) grow histone-DNA co-crystals of defined composition in order to detect the path of the DNA around the octamer and any possible compaction of protein by the DNA, (d) perform comparative structural studies with crystals of biologically relevant histone variants directly,and by molecular dynamics modeling, and (e) perform SAXS and selected hydrodynamic and thermodynamic studies on octamers, core particles, and specifically modified complexes to ascertain the role of solvent and the N-termini of histones on chromatin compaction. We will utilize material from highly differentiated systems (chicken erythrocyte, calf thymus, sea urchin sperm), embryonic systems (sea urchins), and mammalian cells in culture (HL60, HEp-2 & NIH 3T3,+ butyrate, etc.). Ultimately, we want to integrate all this structural information from the normal and """"""""special"""""""" (from transformed cells & embryos) histone octamers and core particles and provide the required linkage between the crystallographic structure and the solution properties of chromatin.
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