Cellular Sticking Can Strongly Reduce Complex Binding by Speeding Dissociation
Cellular Sticking Can Strongly Reduce Complex Binding by Speeding Dissociation
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DOI：
10.1021/acs.jpcb.1c00950
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发表时间：
2021-04-07
影响因子：
3.3
通讯作者：
Gruebele, Martin
中科院分区：
文献类型：
--
作者：
Davis, Caitlin M.;Gruebele, Martin
虽然已经进行了广泛的研究，以确定蛋白质-RNA结合的亲和力，机制和动力学在体外，这样的研究没有考虑到许多弱的非特异性相互作用的细胞充满了潜在的结合伴侣的影响。在这里，我们通过实验测试了细胞环境对蛋白质和RNA在活U-2 OS细胞中的亲和力和结合动力学的作用。我们的模型系统是剪接体蛋白U1 A和它的结合伙伴SL 2的U1 snRNA。结合平衡被扰动的激光诱导的温度跳跃和监测的福斯特共振能量转移。与体外相比，活细胞中的表观结合亲和力降低高达2个数量级。测得的细胞内解离速率系数大2个数量级，而测得的缔合速率系数没有变化。后者不是由于细胞中未复合的U1 A和SL 2的大分子拥挤或非特异性粘附而预期的。一个定量模型符合我们的实验结果，主要的细胞效应是，U1 A和SL 2坚持细胞成分能够结合，只是没有那么强烈的自由复合物。这一观察结果表明，在体外测量或设计的高结合亲和力是必要的适当的结合在体内，其中存在与许多非特异性相互作用的竞争，特别是对于强相互作用的物种与高电荷或大的疏水表面积。
While extensive studies have been carried out to determine protein-RNA binding affinities, mechanisms, and dynamics in vitro, such studies do not take into consideration the effect of the many weak nonspecific interactions in a cell filled with potential binding partners. Here we experimentally tested the role of the cellular environment on affinity and binding dynamics between a protein and RNA in living U-2 OS cells. Our model system is the spliceosomal protein U1A and its binding partner SL2 of the U1 snRNA. The binding equilibrium was perturbed by a laser-induced temperature jump and monitored by Forster resonance energy transfer. The apparent binding affinity in live cells was reduced by up to 2 orders of magnitude compared to in vitro. The measured in-cell dissociation rate coefficients were up to 2 orders of magnitude larger, whereas no change in the measured association rate coefficient was observed. The latter is not what would be anticipated due to macromolecular crowding or nonspecific sticking of the uncomplexed U1A and SL2 in the cell. A quantitative model fits our experimental results, with the major cellular effect being that U1A and SL2 sticking to cellular components are capable of binding, just not as strongly as the free complex. This observation suggests that high binding affinities measured or designed in vitro are necessary for proper binding in vivo, where competition with many nonspecific interactions exists, especially for strongly interacting species with high charge or large hydrophobic surface areas.