Gene V protein dimerization and cooperativity of binding of poly(dA).
Gene V protein dimerization and cooperativity of binding of poly(dA).
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基因 V 蛋白二聚化和聚 (dA) 结合的协同性。
DOI：
10.1021/bi961050c
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发表时间：
1996
期刊：
影响因子：
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通讯作者：
Terwilliger,TC
中科院分区：
文献类型：
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作者：
Terwilliger,TC
噬菌体 f1 的基因 V 蛋白是一种与单链核酸协同结合的二聚体蛋白。为了确定单体-二聚体平衡是否对基因V蛋白与核酸结合的热力学有明显影响，使用尺寸排阻色谱法研究了蛋白质二聚体的解离常数。在浓度范围为 5 × 10-10 至 1.2 × 10-5M 时，蛋白质的斯托克斯半径与基因 V 蛋白二聚体的预期相同。蛋白质的斯托克斯半径也与含有 10 mM Tris-HCl、pH 7.4 和 1 mM EDTA 的缓冲液中从 0.2 到 1.0 M NaCl 的盐浓度无关。使用适用于同时结合两条核酸链的二聚蛋白的蛋白质-蛋白质相互作用的简化晶格模型研究了二聚体基因V蛋白与聚(dA)的结合。使用 Record 等人的理论解释这种二聚体蛋白与核酸结合的盐依赖性 C= [d log(Kintω)]/[d log([NaCl])]。 (Record, M. T., et al. (1976)J.Mol.Biol.107, 145-158)表明C是二聚体而不是单体结合后从蛋白质和核酸释放的阳离子和阴离子数量的函数。通过对结合协同程度敏感的滴定实验研究了基因 V 蛋白与聚 (dA) 结合的协同性。协同因子 ω（定义为与先前结合的二聚体相邻的位点与孤立位点的结合常数之比）被发现对盐相对不敏感，在 3 °C 和 23 °C 下与聚（dA）结合的值在 2000−7000 范围内。这种高协同性因子支持蛋白质-蛋白质接触在超螺旋基因V蛋白-单链核酸复合物的形成中发挥重要作用的建议。
Gene V protein of bacteriophage f1 is a dimeric protein that binds cooperatively to single-stranded nucleic acids. In order to determine whether a monomer−dimer equilibrium has an appreciable effect upon the thermodynamics of gene V protein binding to nucleic acids, the dissociation constant for the protein dimer was investigated using size-exclusion chromatography. At concentrations ranging from 5 × 10-10to 1.2 × 10-5M, the Stokes radius of the protein was that expected of the dimer of the gene V protein. The Stokes radius of the protein was also independent of salt concentration from 0.2 to 1.0 M NaCl in a buffer containing 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA. The binding of the dimeric gene V protein to poly(dA) was studied using a simplified lattice model for protein−protein interactions adapted for use with a dimeric protein that binds simultaneously to two strands of nucleic acid. Interpretation of the salt dependence,C= [d log(Kintω)]/[d log([NaCl])], of binding of such a dimeric protein to nucleic acid using the theory of Record et al. (Record, M. T., et al. (1976)J.Mol.Biol.107, 145−158) indicates thatCis a function of the numbers of cations and anions released from protein and nucleic acid upon binding of the dimer, not of the monomer. Cooperativity of gene V protein binding to poly(dA) was studied with titration experiments that are sensitive to the degree of cooperativity of binding. The cooperativity factor ω, defined as the ratio of the binding constant for a site adjacent to a previously bound dimer to that for an isolated site, was found to be relatively insensitive to salt, with a value in the range of 2000−7000 for binding to poly(dA) at 3 °C and at 23 °C. This high cooperativity factor supports the suggestion that protein−protein contacts play a major role in the formation of the superhelical gene V protein−single-stranded nucleic acid complex.