Selectivity of metal binding and metal-induced stability of Escherichia coli NikR.
NikR from Escherichia coli is a nickel-responsive transcription factor that regulates the expression of a nickel ion transporter. Metal analysis reveals that NikR can bind a variety of divalent transition metals, including Ni(II), Cu(II), Zn(II), Co(II), and Cd(II). The selectivity of metal binding to NikR was investigated by using electronic absorption spectroscopy and small-molecule competitors. The relative affinities, Mn(II) < Co(II) < Ni(II) < Cu(II) > or = Zn(II), follow the Irving-Williams series of metal-complex stabilities. Similar metal affinities were measured for the isolated metal-binding domain of NikR. To determine if any of these metal ions confer a differential effect on NikR, the stability of the metal-bound complexes was examined. In both thermal and chemical denaturation experiments, nickel binding stabilizes the protein more than any of the other metals tested. Thermal denaturation experiments indicate that metal dissociation occurs after loss of secondary structure, but there was no evidence for metal binding to unfolded protein following reversible chemical denaturation. These experiments demonstrate that, although several different metals can bind to NikR, nickel exerts a selective allosteric effect. The implications of these experiments on the in vivo role of NikR as a nickel metalloregulator are discussed.
Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6.
Sheila C
SC
Wang
Alistair V
AV
Dias
Stephanie L
SL
Bloom
Deborah B
DB
Zamble
D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D019216:Metals, Heavy / Q000737:chemistry* / Q000378:metabolism; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis; D013816:Thermodynamics; D014965:X-Rays
D029968:Escherichia coli Proteins; D019216:Metals, Heavy; C402409:NikR protein, E coli; D010446:Peptide Fragments; D012097:Repressor Proteins; D003300:Copper; D009532:Nickel; D019791:Guanidine