NMR spectroscopy of 113Cd(II)-substituted gene 32 protein.
Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. This intrinsic zinc is retained within the DNA-binding core fragment, g32P-(A+B) (residues 22-253), obtained by limited proteolysis of the intact protein. Ultraviolet circular dichroism provides evidence that Zn(II) binding causes significant changes in the conformation of the peptide chain coupled with alterations in the microenvironments of tryptophan and tyrosine side chains. NMR spectroscopy of the 113Cd(II) derivative of g32P-(A+B) at both 44.4 and 110.9 MHz shows a single 113Cd resonance, delta 637, a chemical shift consistent with coordination to three of the four sulfhydryl groups in the protein. In vitro mutagenesis of Cys166 to Ser166 creates a mutant g32P that still contains 1 Zn(II)/molecule. This mutant protein when substituted with 113Cd(II) shows a 113Cd signal with a delta and a line width the same as those observed for the wild-type protein. Thus, the S-ligands to the metal ion appear to be contributed by Cys77, Cys87, and Cys90. Relaxation data suggest that chemical shift anisotropy is the dominant, but not exclusive, mechanism of relaxation of the 113Cd nucleus in g32P, since a dipolar modulation from ligand protons is observed at 44.4 MHz but not at 110.9 MHz. Complexation of core 113Cd g32P with d(pA)6 or Co(II) g32P with poly(dT) shows only minor perturbation of the NMR signal or d-d electronic transitions, respectively, suggesting that the metal ion in g32P does not add a ligand from the bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Giedroc
D P
DP
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510.
Johnson
B A
BA
Armitage
I M
IM
Coleman
J E
JE
D002104:Cadmium / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism*; D007554:Isotopes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D013053:Spectrophotometry; D013604:T-Phages / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification; D015032:Zinc / Q000494:pharmacology
D004268:DNA-Binding Proteins; D007554:Isotopes; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D002104:Cadmium; D015032:Zinc