Event Title

Purification and Characterization of Nickel Uptake Regulator (NUR)

Presenter Information

Denise Peppers, Winthrop University

Faculty Mentor

Nicholas Grossoehme, Ph.D.

College

College of Arts and Sciences

Department

Chemistry, Physics and Geology

Location

DiGiorgio Campus Center, Room 220

Start Date

24-4-2015 3:20 PM

Description

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.

Comments

Presented at the South Carolina TRiO McNair Symposium, June 2014; the SAEOPP McNair/SSS Scholars Research Conference, June 2014; and the Winthrop University Summer Undergraduate Research Experience (SURE) Symposium, July 2014

Supported by a grant from Research Corporation and an NIH-INBRE grant from the National Center for Research Resources and the National Institute of General Medical Sciences

Winner, 1st Place, Physical Sciences Oral Presentations, SAEOPP Conference, June 2014

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Apr 24th, 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.