Zeeb Markus, Max Klaas E A, Weininger Ulrich, Löw Christian, Sticht Heinrich, Balbach Jochen
Laboratorium für Biochemie, Universität Bayreuth, D-95440 Bayreuth, Germany.
Nucleic Acids Res. 2006;34(16):4561-71. doi: 10.1093/nar/gkl376. Epub 2006 Sep 6.
Cold shock proteins (CSP) belong to the family of single-stranded nucleic acid binding proteins with OB-fold. CSP are believed to function as 'RNA chaperones' and during anti-termination. We determined the solution structure of Bs-CspB bound to the single-stranded DNA (ssDNA) fragment heptathymidine (dT7) by NMR spectroscopy. Bs-CspB reveals an almost invariant conformation when bound to dT7 with only minor reorientations in loop beta1-beta2 and beta3-beta4 and of few aromatic side chains involved in base stacking. Binding studies of protein variants and mutated ssDNA demonstrated that Bs-CspB associates with ssDNA at almost diffusion controlled rates and low sequence specificity consistent with its biological function. A variation of the ssDNA affinity is accomplished solely by changes of the dissociation rate. 15N NMR relaxation and H/D exchange experiments revealed that binding of dT7 increases the stability of Bs-CspB and reduces the sub-nanosecond dynamics of the entire protein and especially of loop beta3-beta4.
冷休克蛋白（CSP）属于具有OB折叠的单链核酸结合蛋白家族。CSP被认为作为“RNA伴侣”发挥作用，并参与抗终止过程。我们通过核磁共振光谱法确定了与单链DNA（ssDNA）片段七聚胸苷（dT7）结合的Bs-CspB的溶液结构。当Bs-CspB与dT7结合时，它呈现出几乎不变的构象，仅在β1-β2和β3-β4环以及少数参与碱基堆积的芳香族侧链中有微小的重新定向。对蛋白质变体和突变ssDNA的结合研究表明，Bs-CspB以几乎接近扩散控制的速率与ssDNA结合，且序列特异性较低，这与其生物学功能相符。ssDNA亲和力的变化仅通过解离速率的改变来实现。15N核磁共振弛豫和H/D交换实验表明，dT7的结合增加了Bs-CspB的稳定性，并降低了整个蛋白质尤其是β3-β4环的亚纳秒级动力学。
