The function of zinc in gene 32 protein from T4.
Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II) bound in a tetrahedral complex to -S- ligands, proposed on spectral evidence to include Cys-77, Cys-87, and Cys-90 [Giedroc, D. P., Keating, K. M., Williams, K. R., Konigsberg, W. H., & Coleman, J. E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8452]. The Zn(II) can be completely removed by treatment with the mercurial reagent p-(hydroxymercuri)benzenesulfonate and ethylenediaminetetraacetic acid. The resultant apo-g32P is rapidly digested by trypsin in contrast to the zinc protein which undergoes specific limited proteolysis to yield a resistant DNA-binding core. Rebinding of Zn(II) to the apoprotein restores the same limited susceptibility to proteolysis displayed by the native Zn(II) protein. In the presence of 150 mM NaCl, Zn(II) g32P reduces the melting temperature Tm of poly[d(A-T)] by 47 degrees C, while apo-g32P is unable to melt poly[d(A-T)] at this salt concentration, as the protein thermally unfolds before melting can take place. At 25 mM NaCl, however, apo-g32P lowers the Tm of poly[d(A-T)] by 36 degrees C, but the melting curve is broad compared to the steep cooperative melting induced by Zn(II) g32P. Association constants Ka calculated from the poly[d(A-T)] melting curves for Zn(II) and apo-g32P differ by 3 orders of magnitude, 4.8 X 10(10) M-1 and 4.3 X 10(7) M-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Giedroc
D P
DP
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Keating
K M
KM
Williams
K R
KR
Coleman
J E
JE
D001059:Apoproteins / Q000378:metabolism; D002151:Calorimetry; D002413:Cations, Divalent; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D011067:Poly dA-dT / Q000378:metabolism; D011485:Protein Binding; D013604:T-Phages / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D001059:Apoproteins; D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D014764:Viral Proteins; D011067:Poly dA-dT; D015032:Zinc