Metal-induced structural organization and stabilization of the metalloregulatory protein MntR.
MntR is a metalloregulatory protein that helps to modulate the level of manganese in Bacillus subtilis. MntR shows a metal-response profile distinct from other members of the DtxR family of metalloregulatory proteins, which are generally considered to be iron(II)-activated. As part of an ongoing effort to elucidate the mechanism and metal-selectivity of MntR, several biophysical studies on wild-type MntR and two active site mutants, MntR E99C and MntR D8M, have been performed. Using circular dichroism (CD) spectroscopy, the thermal stability of these proteins has been examined in the presence of various divalent metal ions. Fluorescence intensity measurements of 8-anilino-1-naphthalenesulfonic acid (ANS) were monitored to examine the folding of these proteins in the presence of different metal ions. These experiments indicate that MntR undergoes a significant conformational change upon metal binding that results in stabilization of the protein structure. These studies also show that the MntR D8M active site mutation causes a detrimental effect on the metal-responsiveness of this protein. Fluorescence anisotropy experiments have been performed to quantify the extent of metal-activated DNA binding by these proteins to two different cognate recognition sequences. Binding of MntR and MntR E99C to the mntA cognate sequence closely parallels that of the mntH operator, confirming that the proteins bind both sequences with comparable affinity depending on the activating metal ion. Fluorescence anisotropy experiments on MntR D8M indicate significantly impaired DNA binding, providing additional evidence that MntR D8M is a dysfunctional regulator.
Golynskiy
Misha V
MV
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0358, USA.
Davis
Talib C
TC
Helmann
John D
JD
Cohen
Seth M
SM
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D006358:Hot Temperature; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D019216:Metals, Heavy / Q000737:chemistry* / Q000378:metabolism; D008715:Methionine / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D019216:Metals, Heavy; C433792:MntR protein, bacteria; D012097:Repressor Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D008345:Manganese; C027132:1-anilino-8-naphthalenesulfonate; D001120:Arginine; D008715:Methionine