Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of the Escherichia coli β-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of the β-clamp is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. We determined relative diffusion coefficients of two sliding clamps using fluorescence correlation spectroscopy. Replacing NaCl by KGlu, the primary cytoplasmic salt in E. coli, results in a decrease of the diffusion coefficient of these proteins consistent with the formation of protein assemblies. The UV-VIS spectrum of the β-clamp labeled with tetramethylrhodamine shows the characteristic absorption band of dimers of rhodamine when KGlu is present in the buffer. This suggests that KGlu induces the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with the S. cerevisiae PCNA sliding clamp, suggesting that KGlu effects are not specific to the β-clamp. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly when E. coli is exposed to high concentrations of extracellular solute. Possible biological implications are discussed.

中文翻译：

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谷氨酸钾和甘氨酸甜菜碱诱导 PCNA 和 β 滑动夹的自组装。

滑动夹是增加 DNA 复制效率的寡聚环状蛋白质。Escherichia coli β-clamp 是一种同型二聚体，其稳定性尤为显着。在中等离子强度的缓冲液中，β-clamp 的解离平衡常数约为 10 pM。库仑静电相互作用已被证明有助于这种显着的稳定性。增加分析缓冲液中的 NaCl 浓度会导致二聚体稳定性降低和亚基解离动力学加快，这与简单的电荷筛选模型一致。在这里，我们研究了非库仑离子对滑动夹低聚特性的影响。我们使用荧光相关光谱确定了两个滑动夹具的相对扩散系数。用大肠杆菌中的主要细胞质盐 KGlu 代替 NaCl。大肠杆菌，导致这些蛋白质的扩散系数降低，与蛋白质组装体的形成一致。当 KGlu 存在于缓冲液中时，用四甲基罗丹明标记的 β-clamp 的 UV-VIS 光谱显示了罗丹明二聚体的特征吸收带。这表明 KGlu 诱导了涉及两个或多个面对面堆叠的环的组装的形成。结果可以基于 KGlu 和蛋白质表面上的官能团之间的不利相互作用进行定量解释，这驱动了掩埋暴露表面的生物分子过程。用 S. cerevisiae PCNA 滑动夹获得了类似的结果，表明 KGlu 效应不是特定于 β-夹。甘氨酸甜菜碱也促进钳位结合，一种两性离子化合物，当大肠杆菌暴露于高浓度的细胞外溶质时，会在细胞内积聚。讨论了可能的生物学意义。