Scientific Report - C. David Stout, Assoc. Member, MB
Nucleic Acid and Protein Cystallography
C.D.Stout
G.S.Prasad
J.Nowakowski
S.J.Lloyd
P.J.Shim
N.Kresge
P.Funke
V.Sridhar
This laboratory carries out experimental x-ray crystallography of macromolecules. Fundamental questions are addressed through structure determination of key molecules involved in biological processes. The research often involves collaboration with faculty at Scripps. The experiments entail biochemical preparation, crystallization and collection and analysis of x-ray diffraction data. Once a structure is solved, experiments are designed to study relationships between structure and function. These entail design and preparation of site-directed mutants and ligand complexes, structure analysis and assays of biological function. Projects focused on iron-sulfur enzymes, fertilization proteins, an integral membrane proton pump, nucleic acid four-way junctions, and RNA-protein complexes have seen significant progress in the past year.
The iron-sulfur enzyme aconitase is used as a molecular laboratory in which to carry out [Fe-S] cluster engineering experiments, in affiliation with the Metalloprotein Structure and Design project at Scripps. The experiments investigate the structural and chemical requirements for the biosynthesis of [Fe-S] clusters in proteins, and are relevant to functional transformations that occur in many [Fe-S] proteins. In collaboration with B.K. Burgess, the properties of [Fe-S] clusters are being probed using a 7Fe ferredoxin from Azotobacter vinelandii as a model system. High-resolution structure analysis has provided a basis for modeling the redox-coupled proton transfer that occurs in this [Fe-S] protein.
An on-going project entails the study of sperm-egg interaction at the molecular level. Four structures of proteins from red and green abalone sperm have been determined at high resolution in collaboration with V.D. Vacquier: the green 16K lysin dimer, the green 18K protein, the red 16K lysin monomer and the red 16K lysin dimer. These proteins dissolve the egg vitelline envelope by a non-enzymatic mechanism during fertilization. The structures afford important insight into the basis of the species-specific interaction of gametes, and lead to a model for the interaction of lysin with its egg receptor.
Experiments with mitochondrial transhydrogenase, in collaboration with Y. Hatefi and G.S. Prasad, have yielded a structure of the extramembranous NADP(H) binding domain. Structure determination of the other extramembranous domain, which binds NAD(H), is in progress. This enzyme provides an excellent model system in which to study the mechanism of proton translocation across membranes (please refer to the report in this volume by Y. Hatefi).
In collaboration with G.F. Joyce, structural analysis of nucleic acid four-way junctions is being done using 2:2 complexes of a DNA enzyme with its RNA substrate. Four-way junctions occur during genetic recombination, and are present in the hairpin ribozyme and spliceosomal RNA. The first crystal structure provided an atomic resolution model of the junction in the ëstacked-Xí conformation. A second crystal structure provides a detailed model for the ëcrossedí conformation, and by comparison to the first reveals the factors that stabilize the two very different junction conformers. These factors underlie the intrinsic, and biologically relevant, flexibility of four-way junctions.
Crystallographic analysis of a 70 kD ribosomal RNA-protein complex has been carried out in collaboration with J.R. Williamson. The complex contains 104 nucleotides from the central domain of 16S rRNA and the ribosomal proteins S6, S15 and S18. The RNA contains two three-helix junctions arranged at opposite ends of a central helix. The S15 protein binds to one of these junctions and a purine-rich internal loop in the central helix; the S6 and S18 proteins bind as a heterodimer to the other three-helix junction. The structure reveals many details of RNA-RNA and RNA-protein interactions, and in combination with biochemical data, provides significant insight into the mechanism of assembly of the 30S subunit of the ribosome (please refer to the report in this volume by J.R. Williamson).
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