Showcase of Undergraduate Research and Creative Endeavors (SOURCE)

Testing the model of Nur function using non-standard amino acids

Nickel uptake regulator (Nur) regulates cellular concentrations of nickel in S. coelicolor and is the only Ni-sensing member of the Fur family of metalloregulators. Understanding Nur is paramount to understanding how the Fur family has evolved to serve diverse functions. Nur is a symmetric homodimer with each monomer containing two metal binding sites: one M-site and one Ni-site. Previous literature indicates the Ni-site as the regulatory site for Nur, however research in the Grossoehme lab has evidence that suggests the M-site as the regulatory site. The proposed regulatory model suggests when the M-site is devoid of metal, the linker between the dimerization domain and the DNA-binding domain is flexible, leaving Nur in an open conformation with low affinity for DNA. Conversely, when metal is bound to the M-site the flexible linker rigidifies, placing Nur in a closed conformation and increasing DNA affinity. 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 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, p-azido-phenylalanine is incorporated into Nur at specific locations using the amber stop codon strategy. Click chemistry will be utilized to covalently attach alkyne-modified FRET or paramagnetic probes. Currently, modified Nur has been expressed and purification is underway.