The broad long term objectives of this research project are to obtain information at the molecular level about protein-nucleic acid interaction. Of particular importance is the furthering of our understanding of factors influencing nucleic acid sequence-selectivity, and the stability of specific complexes. The interaction between proteins and nucleic acids is a fundamental component of cellular processes, as well as being essential for the assembly and functioning of infectious agents such as mammalian retroviruses. The major specific aim of the proposed research is the study of nucleic acid complexes of the nucleocapsid proteins of type 1 human immunodeficiency virus (an infectious agent leading to AIDS), HIV-1 NC p7, and of simian immunodeficiency virus, SIV NC p8. Along with all known retroviral NC proteins, p7 and p8 contain zinc finger CCHC arrays that include highly conserved aromatic residues such as trp and phe. These Zn finger arrays have been implicated in specific nucleic acid binding and packaging. Our research will focus on the use of optical detection of triplet state magnetic resonance (ODMR) of p7 and p8 complexes with heavy atom-derivatized nucleic acids to determine whether aromatic stacking interactions are involved in binding. The triplet state zero-field splitting shifts induced in trp by complex formation with a variety of nucleic acids will be used to quantitatively evaluate the contribution of aromatic stacking interactions to complex stability, and thereby, their influence on sequence selectivity. This goal will require further investigations of echinomycin (a DNA bis-intercalating antibiotic) analogs by ODMR spectroscopy as models of stacking-induced sequence selectivity. In related work, complexes of sequence-specific DNA binding proteins, E. coli trp repressor, lac repressor and Eco RI methyl transferase with 2- thiouracil- or 2-thiothymine-containing oligomers will be studied. Triplet-triplet energy transfer from the sulfur-derivatized base of DNA to protein trp residues will be utilized to obtain structural information. Extensive use will be made of mutated proteins and enzymes that involve conservative substitution of intrinsic trp residues by other amino acids as an aid in interpretation of the spectroscopic results. Any information that we can provide that bears on the origins of sequence selectivity of p7 and p8, in particular, would have important implications in such areas as the design of novel HIV antiviral agents.
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