PECASE: A Difference Infrared Spectroscopic Study of a Nucleotide Binding Protein

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9733566 MacDonald This CAREER plan integrates research and educational components and involves undergraduate chemistry and biology majors as well as high school teachers in all aspects of the project. The goal of the research is to gain further insight into the regulations and mechanism of a protein involved in DNA repair and recombination. Fourier transform infrared (FTIR) difference spectroscopy will be used to investigate the relationship between protein structure and function in the Escherichia coli protein, RecA. These studies should result in a greater understanding of all allosteric changes in RecA that are responsible for regulating DNA strand exchange, a process crucial to DNA recombination and repair. RecA function is regulated by nucleotide binding. Initial studies will focus on the binding of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to RecA. Vibrational spectroscopy will be used to identify specific changes in protein structure induced by the binding of ADP (RecA conformation with decreased affinity for DNA), and ATP (RecA conformation with a higher affinity for DNA). These studies will be performed under a variety of conditions in order to investigate how nucleotide binding affects protein structure in both the active and inactive RecA nucleoprotein filaments. These studies will provide the groundwork for future RecA studies such as (1) how the previously studied changes in RecA structure are affected by other proteins known to interact with RecA in vitro and (2) time-resolved studies that follow nucleotide binding and strand exchange in real time. High school teachers, as well as James Madison University undergraduates in chemistry and biology ,and secondary education minors, will play instrumental roles in both the educational and research aspects of this CAREER grant. A larger number of participants can be involved in research efforts by providing two types of projects (1) biochemical and difference infrared studies of RecA (2) research effor ts that utilize departmental equipment and can be incorporated into the new project oriented biochemistry laboratory. Students involved will gain valuable research experience, learn current biochemical and spectroscopic techniques, and have a greater understanding of the scientific method. The undergraduate participants will contribute to a greater understanding of structure-function relationships in RecA and an exciting new biochemical laboratory. Including current and future high school teachers in this project will ultimately help to attract the next generation of scientists.