The nickel uptake regulator (NUR) is a metalloregulatory protein found in the microorganism Streptomyces coelicolor. It is the first Ni(II)-sensing member of the FUR family of metalloregulatory proteins. NUR is responsible for regulation of a variety of genes involved in nickel uptake and oxidative stress. Interestingly, NUR is also responsible for regulation of the enzyme superoxide dismutase (SOD) within S.coelicolor; it regulates Fe-containing SOD through a direct mechanism and indirectly controls Ni-containing SOD. The goal of this research is to purify and characterize the metal and DNA binding affinities in wild type (WT) NUR. There are two metal binding sites within NUR that are believed to contribute to the function of this protein. The “M-site”, which corresponds to a well-conserved site within FUR proteins, contains Zn(II) in a square-planar geometry when purified from E. coli. The second site, denoted the “Ni-site”, is a unique site in FUR proteins and has been suggested to be the sensory Ni(II)-binding site. The work presented in this poster will describe a series of biophysical experiments that aim to assess the role of each metal site in NUR function. This was approached using a complement of biophysical techniques including site-directed mutagenesis, competitive metal binding assays, fluorescence anisotropy, atomic absorbance spectroscopy, and quantitative chromatography. Together, the data suggests that the “M-site” plays a more profound role in the metal sensory function of NUR.

Purification and characterization of wild type nickel uptake regulator (NUR) from Streptomyces coelicolor

249th National Meeting (2015)

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