Krauss G, Sindermann H, Schomburg U, Maass G
Biochemistry. 1981 Sep 1;20(18):5346-52. doi: 10.1021/bi00521a040.
The complex formation between the single-strand DNA binding protein (ssB protein) from Escherichia coli and oligonucleotides and single-stranded DNA has been studied by using fluorescence titrations, ultracentrifugation measurements, and fast kinetic techniques. Determination of the stoichiometries of oligo(dT)--ssB complexes shows that each of the four subunits of the ssB protein represents a binding site for an oligonucleotide about eight residues long. Occupation of all four binding sites with oligo(dT) or poly(dT) leads to 80% quenching of the intrinsic protein fluorescence. The binding sites are nearly equivalent and independent. For d(pT)16, the intrinsic binding constant is 6 X 10(5) M-1, and for d(pT)30-40, which is long enough to extend continuously over the ssB tetramer, the binding constant is higher than 5 X 10(8) M-1. Oligoadenylates bind about 2 orders of magnitude weaker than the corresponding oligo(dT) species. The binding of oligo(dT) is very weakly dependent on ionic strength, in contrast to the oligo(dA)--ssB complex formation. For d(pT)8, d(pT)16, and d(pT)30-40, the complex formation can be described by a simple one-step reaction. The strength of the interaction is mainly expressed in the rate constant of dissociation. In the cooperative complexes with poly(dT) or poly(dA), all four binding sites on the ssB tetramer are also occupied. It is concluded that single-stranded DNA is coiled around the ssB molecule. Fluorescence melting experiments of the complexes show that the conformation of the single-stranded DNA has a strong influence on the stability of the complexes.
通过荧光滴定、超速离心测量和快速动力学技术，研究了来自大肠杆菌的单链DNA结合蛋白（ssB蛋白）与寡核苷酸和单链DNA之间的复合物形成。寡聚（dT）-ssB复合物化学计量的测定表明，ssB蛋白的四个亚基中的每一个都代表一个约八个残基长的寡核苷酸的结合位点。用寡聚（dT）或聚（dT）占据所有四个结合位点会导致蛋白质固有荧光淬灭80%。这些结合位点几乎是等效且独立的。对于d（pT）16，固有结合常数为6×10⁵ M⁻¹，对于d（pT）30 - 40，其长度足以连续延伸覆盖ssB四聚体，结合常数高于5×10⁸ M⁻¹。寡聚腺苷酸的结合比相应的寡聚（dT）物种弱约2个数量级。与寡聚（dA）-ssB复合物形成相反，寡聚（dT）的结合对离子强度的依赖性非常弱。对于d（pT）8、d（pT）16和d（pT）30 - 40，复合物形成可以用简单的一步反应来描述。相互作用的强度主要表现在解离速率常数上。在与聚（dT）或聚（dA）的协同复合物中，ssB四聚体上的所有四个结合位点也被占据。得出的结论是，单链DNA缠绕在ssB分子周围。复合物的荧光熔解实验表明，单链DNA的构象对复合物的稳定性有很大影响。
