Showcase of Undergraduate Research and Creative Endeavors (SOURCE)

Testing the Model of NUR Function: Incorporation of non-Standard Amino Acids

Nur is a unique member of the Fur family of metalloregulators, in that it is the only Ni-sensing member of the family and, to date, is only present in the bacterium Streptomyces coelicolor. Understanding Nur is paramount to understanding how the Fur family has evolved to serve diverse functions. Nur is a symmetric homodimer with monomers bound at the dimerization domain. Each monomer contains two metal binding sites: one M-site and one Ni-site. Previous literature indicates that the Ni-site operate as the regulatory site for Nur, however research in the Grossoehme lab has evidence that suggests the M-site as the regulatory site. This project proposes to use EPR and FRET as complimentary techniques to test the working model of regulation. This will be accomplished by attaching probes at the specific sites in the protein structure. The magnetic interactions of the EPR probe will allow studies on movement of the flexible linker, while large conformation changes will be detectable by the relative distance between the FRET probes. To ensure that the probes are only attached where desired, Nur must contain a completely unique amino acid at the correct locations, making incorporation of non-standard amino acids necessary. In this project, the nur gene is cloned into an expression plasmid and mutated at the desired locations. Transformation into the genetically reprogrammed organism needed for non-standard amino acid incorporation and purification of mutated Nur is underway.