CONFORMATION-DEPENDENT CLEAVAGE OF STAPHYLOCOCCAL NUCLEASE WITH A DISULFIDE-LINKED IRON CHELATE
CONFORMATION-DEPENDENT CLEAVAGE OF STAPHYLOCOCCAL NUCLEASE WITH A DISULFIDE-LINKED IRON CHELATE
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DOI：
10.1073/pnas.89.14.6383
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发表时间：
1992-07-15
影响因子：
11.1
通讯作者：
FOX, RO
中科院分区：
文献类型：
--
作者：
ERMACORA, MR;DELFINO, JM;FOX, RO
本文报道了（EDTA-2-氨乙基）2-吡啶基二硫化物的合成和评价。通过使用这种容易制备的半胱氨酸特异性亲水性试剂，乙二胺三乙酸-Fe 3+络合物（EDTA-Fe）共价连接到葡萄球菌核酸酶中的单个基因工程半胱氨酸残基。在添加铁还原剂抗坏血酸后，核酸酶-EDTA-Fe缀合物经历由活性氧介导的蛋白质自切割反应。产物的序列分析表明，切割发生在三级结构的EDTA-Fe附着位点附近。在变性剂的存在下，切割模式发生变化，反应仅限于半胱氨酸连接位点序列近端的残基。这些结果表明，由EDTA-Fe介导的分子内蛋白质切割反应可用于评估蛋白质构象的变化。所描述的试剂应该是一个有用的工具，在结构映射的非天然蛋白质状态的平衡，如熔融球，这是经常难治性的传统结构分析填充。
We report the synthesis and evaluation of (EDTA-2-aminoethyl) 2-pyridyl disulfide. By using this easily prepared cysteine-specific hydrophilic reagent, an ethylenediaminetriacetic acid-Fe3+ complex (EDTA-Fe) was covalently attached to a single genetically engineered cysteine residue in staphylococcal nuclease. Upon addition of the iron reductant ascorbate, the nuclease-EDTA-Fe conjugate underwent a protein self-cleavage reaction mediated by reactive oxygen species. Sequence analysis of the products indicated that cleavage occurs close in tertiary structure to the EDTA-Fe attachment site. In the presence of denaturants, the cleavage pattern changes and the reaction is limited to residues proximal in sequence to the cysteine attachment site. These results indicate that intramolecular protein cleavage reactions mediated by EDTA-Fe can be used to evaluate changes in protein conformation. The reagent described should be a useful tool in the structural mapping of nonnative protein states populated at equilibrium, such as the molten globule, that are frequently refractory to conventional structure analysis.