Purification and Characterization of Nickel Uptake Regulator (NUR)
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Faculty Mentor
Nicholas Grossoehme, Ph.D.
Abstract
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 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, our data suggest that the “M-site” plays a more profound role in the metal sensory function of NUR.
Start Date
24-4-2015 3:20 PM
Purification and Characterization of Nickel Uptake Regulator (NUR)
DiGiorgio Campus Center, Room 220
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 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, our data suggest that the “M-site” plays a more profound role in the metal sensory function of NUR.
