1H NMR studies of T4 gene 32 protein: effects of zinc removal and reconstitution.
Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol bound in a tetrahedral ligand field. 113Cd NMR studies of Cd-substituted wild-type and mutant (Cys166----Ser166) g32Ps show Cys77, Cys87, and Cys90 to provide three sulfur donor atoms as ligands to the metal ion [Giedroc, D. P., Johnson, B. A., Armitage, I. M., & Coleman, J. E. (1989) Biochemistry 28, 2410]. Proton NMR signals from the His and Trp side chains of the protein have been followed as a function of pH and metal ion removal by biosynthesizing the protein with amino acids carrying protons at specific positions in a background of perdeuteriated aromatic amino acids. Only one of the two pairs of His resonances (from His64 and His81) titrates over the pH range 8.0-5.9. The nontitrating His side chain is most likely ligated to the metal ion. Upon Zn(II) removal, 1H NMR spectra of the fully protonated g32P-(A + B) exhibit substantial signal broadening in several regions of the spectrum, while the His 2,4-1H resonances are broadened beyond detection. The 1H NMR spectral characteristics of the original protein are restored by reconstitution with stoichiometric Zn(II). The broadening of the 1H NMR signals is not due to oligomerization of the protein, since small-angle X-ray scattering experiments show that the average radius of gyration of the apo-g32P-(A + B) is 25.0 A and that of the reconstituted Zn(II)-g32P-(A + B) is 31.2 A.(ABSTRACT TRUNCATED AT 250 WORDS)
Pan
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Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510.
Giedroc
D P
DP
Coleman
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JE
D004268:DNA-Binding Proteins; D006639:Histidine; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D014364:Tryptophan; D014764:Viral Proteins; D015032:Zinc
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D006639:Histidine; D014364:Tryptophan; D015032:Zinc