This project will explore the molecular basis of ssDNA recognition by the ssDNA binding proteins through intensive biophysical study of Ff gene 5 protein, a prototypical member of the family. This particular class of protein-nucleic acid interaction is much less well characterized than protein-dsDNA systems. A detailed description of the molecular bases of ssDNA binding an understanding will provide an essential underprinting for future attempts to affect disease states by alteration of genetic controls. As a closely-related goal, structural study of gene 5 protein will also contribute to an understanding of the fundamental processes governing the folding and stability of proteins. The research program will focus heavily on defining the solution conformation and dynamics of gene 5 protein and its interaction with ssDNA through extensive 1H, 13C, and 15N-NMR experiments. Site-directed mutant species will be used to separate the DNA-binding and cooperative assembly components of its activity for ready experimental manipulation. Complete assignment of the 1H, 13C and 15N-spectra of gene 5 protein and its complex with a tight-binding oligonucleotide will be undertaken. The solution conformations of these species, with emphasis on the functionally critical DNA Binding Wing, will be determined with NMR-based distance geometry and restrained molecular dynamics methods. Heteronuclear relaxation experiments will investigate dynamic conformational processes and changes in dynamics which accompany oligonucleotide binding. Information on dynamic processes obtained from these experiments will be incorporated into NMR structure determination algorithms. The detailed model of the solution conformation of gene 5 protein resulting from the structure determination will provide a datum for interpreting structural and dynamic changes exhibited by mutant protein species with unnatural folding or stability properties. Assembly of the supermolecular gene 5 protein-ssDNA complex will be modelled with computer graphics procedures and the resulting models tested by biophysical examination of mutant protein ssDNA binding activity.
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| Michnicka, M J; Harper, J W; King, G C (1993) Selective isotopic enrichment of synthetic RNA: application to the HIV-1 TAR element. Biochemistry 32:395-400 |
