Potassium Glutamate and Glycine Betaine Induce Self-Assembly of the PCNA and β-Sliding Clamps
Potassium Glutamate and Glycine Betaine Induce Self-Assembly of the PCNA and β-Sliding Clamps
复制标题
谷氨酸钾和甘氨酸甜菜碱诱导 PCNA 和 β 滑动夹的自组装
DOI：
10.1016/j.bpj.2020.11.013
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发表时间：
2021
影响因子：
3.4
通讯作者：
Levitus, Marcia
中科院分区：
文献类型：
--
作者：
Purohit, Anirban;Douma, Lauren G.;Bloom, Linda B.;Levitus, Marcia
滑动夹是一种寡聚环状蛋白质，可以提高DNA复制的效率。大肠杆菌的β-夹，一种同源二聚体，其稳定性特别显著。在中等离子强度的缓冲液中，β-钳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。增加测定缓冲液中的NaCl浓度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，我们研究非库仑离子对滑动钳的低聚性能的影响。我们用荧光相关光谱法测定了两个滑动夹的相对扩散系数。用KGlu（大肠杆菌中的主要胞质盐）代替NaCl，导致这些蛋白质的扩散系数降低，这与蛋白质组装体的形成一致。当KGlu存在于缓冲液中时，四甲基罗丹明标记的β-钳的紫外-可见光谱显示罗丹明二聚体的特征吸收带。这表明KGlu诱导了涉及两个或更多个面对面堆叠的环的组装体的形成。结果可以定量解释的基础上KGlu和蛋白质表面上的官能团，这驱动生物分子过程，掩埋暴露的表面之间的不利相互作用。用酵母PCNA滑动钳也得到了类似的结果，表明KGlu的作用不是β-钳所特有的。甘氨酸甜菜碱也能促进钳位结合，甘氨酸甜菜碱是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，它会在细胞内积累。可能的生物影响进行了讨论。
Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of theEscherichia coli β-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of theβ-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 inE. coli, results in a decrease of the diffusion coefficient of these proteins consistent with the formation of protein assemblies. The UV-vis spectrum of theβ-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 theSaccharomyces cerevisiaePCNA sliding clamp, suggesting that KGlu effects are not specific to theβ-clamp. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly whenE. coliis exposed to high concentrations of extracellular solute. Possible biological implications are discussed.