Ebright Y W, Chen Y, Pendergrast P S, Ebright R H
Department of Chemistry, Rutgers University, New Brunswick, New Jersey 08855.
Biochemistry. 1992 Nov 10;31(44):10664-70. doi: 10.1021/bi00159a004.
We have developed a simple procedure to incorporate an EDTA-metal complex at a rationally selected site within a full-length protein. Our procedure has two steps: In step 1, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step 2, we derivatize the resulting protein with S-(2-pyridylthio)cysteaminyl-EDTA-metal, a novel aromatic disulfide derivative of EDTA-metal. We have used this procedure to incorporate an EDTA-iron complex at amino acid 2 of the helix-turn-helix motif of each of two helix-turn-helix motif sequence-specific DNA binding proteins, catabolite gene activator protein (CAP) and Cro, and we have analyzed EDTA-iron-mediated DNA affinity cleavage by the resulting protein derivatives. The CAP derivative cleaves DNA at base pair 2 of the DNA half-site in the protein-DNA complex, and the Cro derivative cleaves DNA at base pairs -3 to 5 of the DNA half-site in the protein-DNA complex. We infer that amino acid 2 of the helix-turn-helix motif of CAP is close to base pair 2 of the DNA half-site in the CAP-DNA complex in solution and that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs -3 to 5 of the DNA half-site in the Cro-DNA complex in solution.(ABSTRACT TRUNCATED AT 250 WORDS)
我们开发了一种简单的方法，可将乙二胺四乙酸（EDTA）-金属络合物掺入全长蛋白质中合理选择的位点。我们的方法有两个步骤：第一步，我们使用定点诱变在目标位点引入一个独特的溶剂可及半胱氨酸残基。第二步，我们用S-（2-吡啶硫基）半胱氨酰-EDTA-金属对所得蛋白质进行衍生化，这是一种新型的EDTA-金属芳族二硫化物衍生物。我们已使用此方法将EDTA-铁络合物掺入两种螺旋-转角-螺旋基序序列特异性DNA结合蛋白（分解代谢基因激活蛋白（CAP）和Cro）的每个螺旋-转角-螺旋基序的第2位氨基酸处，并且我们分析了所得蛋白质衍生物的EDTA-铁介导的DNA亲和切割。CAP衍生物在蛋白质-DNA复合物中DNA半位点的第2个碱基对处切割DNA，而Cro衍生物在蛋白质-DNA复合物中DNA半位点的第-3至5个碱基对处切割DNA。我们推断，在溶液中，CAP的螺旋-转角-螺旋基序的第2位氨基酸靠近CAP-DNA复合物中DNA半位点的第2个碱基对，并且Cro的螺旋-转角-螺旋基序的第2位氨基酸靠近溶液中Cro-DNA复合物中DNA半位点的第-3至5个碱基对。（摘要截断于250字）
