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)