The long-term objective of this research program is to acquire detailed information regarding protein-nucleic acid interactions at a molecular level. These interactions are of fundamental importance in cellular metabolism, and knowledge in this area may be of great benefit to, and foster applications in the health area. Use will be made of optical detection of triplet state magnetic resonance (ODMR) spectroscopy with which we will study mainly the tryptophan residues of nucleic acid-binding proteins. Interactions between Trp residues and nucleic acids will be evaluated using ODMR. Use will be made of the external heavy atom effect induced by close interactions between Trp residues and heavy atom- derivatized nucleic acids. In addition, effects on the excited states of Trp which result from stacking interactions with underivatized nucleic acid bases will be investigated by ODMR. We propose to study the complexing of proteins which bind preferentially to single-stranded nucleic acids (SSB proteins) with polynucleotides and oligonucleotides. Once the existence of aromatic stacking interactions with wild type SSBs has been established, we will study the complexes formed by mutant proteins to identify specific Trp residues which are responsible for stabilization of the complex through stacking interactions. Site- selected oligonucleotide mutagenesis will be employed to form mutants of E. coli SSB and of T4 gene 32 protein, whose genes have been cloned by one of our collaborators. Fluorescence and salt- back titrations will be used, as well, to evaluate the effects of mutations on the stability of the nucleic acid complexes. We will use a strategy similar to that which we have employed recently to identify the stacked Trp residues in E. coli SSB. Other mutants of gene 32 protein such as an amber mutant, will be studied as they are available. In addition to using heavy atom-derivatized nucleic acids, we will begin to employ poly (s2U) as a substrate. Using triplet-triplet energy transfer from this substrate, we can produce the excited states of only those Trp which are in the vicinity of (and stacked with) the bases. Conservation of spin alignment during energy transfer will enable us to obtain structural data on the Trp-base stacked complex. In addition to continuing our work on mutant E. coli SSBs and T4 gene 32 proteins, we also will investigate the DNA binding protein_from the filamentous phage Pfl. We will now begin to make measurements on eukaryotic SSBs, such as UP1 and UP2 from calf thymus, and the p10 protein from Rauscher murine leukemia virus. Preliminary work on the latter protein is complete.
Showing the most recent 10 out of 39 publications
