Giedroc D P, Keating K M, Williams K R, Coleman J E
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Biochemistry. 1987 Aug 25;26(17):5251-9. doi: 10.1021/bi00391a007.
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)
基因32蛋白（g32P）是来自噬菌体T4的单链DNA结合蛋白，含有1摩尔以四面体络合物形式与-S-配体结合的Zn（II），根据光谱证据推测其中包括半胱氨酸-77、半胱氨酸-87和半胱氨酸-90 [吉德罗克，D.P.，基廷，K.M.，威廉姆斯，K.R.，科尼格斯伯格，W.H.，& 科尔曼，J.E.（1986年）《美国国家科学院院刊》83，8452]。用汞试剂对-（羟基汞）苯磺酸盐和乙二胺四乙酸处理可将Zn（II）完全去除。与锌蛋白相比，所得的脱辅基g32P会被胰蛋白酶迅速消化，锌蛋白会经历特定的有限蛋白水解以产生有抗性的DNA结合核心。Zn（II）与脱辅基蛋白的重新结合恢复了天然Zn（II）蛋白所显示的相同有限的蛋白水解敏感性。在150 mM氯化钠存在的情况下，Zn（II）g32P使聚[d（A-T）]的解链温度Tm降低47摄氏度，而脱辅基g32P在此盐浓度下无法使聚[d（A-T）]解链，因为蛋白质在解链发生之前就发生了热变性。然而，在25 mM氯化钠时，脱辅基g32P使聚[d（A-T）]的Tm降低36摄氏度，但与Zn（II）g32P诱导的陡峭协同解链相比，解链曲线较宽。根据聚[d（A-T）]解链曲线计算的Zn（II）和脱辅基g32P的缔合常数Ka相差3个数量级，分别为4.8×10¹⁰ M⁻¹和4.3×10⁷ M⁻¹。（摘要截断于250字）
