Binding mode transitions of Escherichia coli single strand binding protein-single-stranded DNA complexes. Cation, anion, pH, and binding density effects.
Binding mode transitions of Escherichia coli single strand binding protein-single-stranded DNA complexes. Cation, anion, pH, and binding density effects.
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DOI：
10.1016/s0021-9258(18)68829-5
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发表时间：
1988-04
期刊：
影响因子：
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通讯作者：
W. Bujalowski;Leslie B. Overman;T. Lohman
中科院分区：
文献类型：
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作者：
W. Bujalowski;Leslie B. Overman;T. Lohman
我们扩展了对大肠杆菌单链结合（SSB）蛋白和单链DNA之间形成的多种结合模式的研究（Lohman，T. M. & Overman，L. B。（1985）J.Biol.Chem.260，3594-3603; Bujalowski，W. & Lohman，T. M.（1986）Biochemistry 25，7799-7802），通过检查阴离子、pH、BaCl 2和蛋白质结合密度对这些结合模式之间的转变的影响。“反向”滴定，监测淬灭的SSB蛋白质的内在色氨酸荧光后，加入聚（dT）已被用来测量的表观位点大小的复杂的在25摄氏度，pH值8.1和6.9的函数氟化钠，氯化钠，溴化钠，和氯化镁浓度。在进行“反向”滴定的所有条件下，我们观察到三种不同的结合模式，位点大小为35 +/-2，56 +/-3和65 +/-3个核苷酸/SSB四聚体;然而，三种结合模式之间的转换强烈地依赖于阳离子和阴离子的价态，类型，阳离子和阴离子的净吸收伴随着在pH 6.9下从（SSB）35到（SSB）56结合模式的转变，而在pH 8.1下，这种转变是不依赖于阴离子的，并且仅发生阳离子的净吸收。从（SSB）56到（SSB）65结合模式的转变取决于pH 6.9和8.1（25 ℃）下的阳离子和阴离子，并且阳离子和阴离子的净吸收伴随着这种转变。我们还通过监测SSB蛋白-聚（dT）复合物的沉降系数随MgCl 2浓度（20 ℃，pH 8.1）的变化来检查过渡，并观察到s20，w的增加，这与复合物的表观位点大小的增加相一致，如通过荧光滴定所测量的。复合物的摩擦系数在从（SSB）35到（SSB）65结合模式的过程中降低了2倍，表明复合物在整个过渡过程中逐渐压实。（SSB）35和（SSB）56复合物之间的转换取决于蛋白质结合密度，较低的位点大小（SSB）35复合物有利于较高的结合密度。这些结果表明，各种SSB蛋白质-单链DNA结合模式之间的转换是复杂的过程，取决于一些解决方案的变量，是相互联系的。（400字处截断摘要）
We have extended our investigations of the multiple binding modes that form between the Escherichia coli single strand binding (SSB) protein and single-stranded DNA (Lohman, T. M. & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W. & Lohman, T. M. (1986) Biochemistry 25, 7799-7802) by examining the effects of anions, pH, BaCl2, and protein binding density on the transitions among these binding modes. “Reverse” titrations that monitor the quenching of the intrinsic tryptophan fluorescence of the SSB protein upon addition of poly(dT) have been used to measure the apparent site size of the complex at 25 degrees C in pH 8.1 and 6.9 as a function of NaF, NaCl, NaBr, and MgCl2 concentrations. Under all conditions in which “reverse” titrations were performed, we observe three distinct binding modes with site sizes of 35 +/- 2, 56 +/- 3, and 65 +/- 3 nucleotides/SSB tetramer; however, the transitions among the three binding modes are strongly dependent upon both the cation and anion valence, type, and concentration as well as the pH. A net uptake of both cations and anions accompanies the transitions from the (SSB)35 to the (SSB)56 binding mode at pH 6.9, whereas at pH 8.1 this transition is anion-independent, and only a net uptake of cations occurs. The transition from the (SSB)56 to the (SSB)65 binding mode is dependent upon both cations and anions at both pH 6.9 and 8.1 (25 degrees C), and a net uptake of both cations and anions accompanies this transition. We have also examined the transitions by monitoring the change in the sedimentation coefficient of the SSB protein-poly(dT) complex as a function of MgCl2 concentration (20 degrees C, pH 8.1) and observe an increase in s20,w, which coincides with the increase in apparent site size of the complex, as measured by fluorescence titrations. The frictional coefficient of the complex decreases by a factor of two in progressing from the (SSB)35 to the (SSB)65 binding mode, indicating a progressive compaction of the complex throughout the transition. The transition between the (SSB)35 and the (SSB)56 complex is dependent on the protein binding density, with the lower site size (SSB)35 complex favored at higher binding density. These results indicate that the transitions among the various SSB protein-single-stranded DNA binding modes are complex processes that depend on a number of solution variables that are thermodynamically linked.(ABSTRACT TRUNCATED AT 400 WORDS)