JILA, University of Colorado Boulder and National Institute of Standards and Technology, Boulder, Colorado 80309, United States.
Department of Chemistry, University of Colorado Boulder, Boulder, Colorado 80309, United States.
J Phys Chem B. 2023 Mar 23;127(11):2430-2440. doi: 10.1021/acs.jpcb.3c00245. Epub 2023 Mar 14.
Functionality in many biological systems, including proteins and nucleic acid structures, including protein and nucleic acid riboswitch structures, can depend on cooperative kinetic behavior between multiple small molecule ligands. In this work, single-molecule FRET data on the lysine riboswitch reveals that affinity for the cognate lysine ligand increases significantly with K, providing evidence for synergism between lysine/K binding to the aptamer and successful folding of the riboswitch. To describe/interpret this more complex kinetic scenario, we explore the conventional 4-state ("square") model for aptamer binding as a function of K. Extension into this additional dimension generates a novel "cube" model for riboswitch folding dynamics with respect to lysine/K binding, revealing that riboswitch folding () and unfolding () rate constants increase and decrease dramatically with K, respectively. Furthermore, temperature-dependent single-molecule kinetic studies indicate that the presence of K entropically enhances the transition state barrier to folding but partially compensates for this by increasing the overall exothermicity for lysine binding. We rationalize this behavior as evidence that K facilitates hydrogen bonding between the negatively charged carboxyl group of lysine and the RNA, increasing structural rigidity and lowering entropy in the binding pocket. Finally, we explore the effects of cation size with Na and Cs studies to demonstrate that K is optimally suited for bridging interactions between lysine and the riboswitch aptamer domain. Regulation of lysine production and transport, dictated by the riboswitch's ability to recognize and bind lysine, is therefore intimately tied to the presence of K in the binding pocket and is strongly modulated by local cation conditions. The results suggest an increase in lysine riboswitch functionality by sensitivity to additional species in the cellular riboswitch environment.
在许多生物系统中，包括蛋白质和核酸结构，包括蛋白质和核酸核糖开关结构，功能可能依赖于多个小分子配体之间的协同动力学行为。在这项工作中，赖氨酸核糖开关的单分子 FRET 数据表明，对同源赖氨酸配体的亲和力随 K 的增加而显著增加，这为赖氨酸/K 与适体的结合和核糖开关的成功折叠之间的协同作用提供了证据。为了描述/解释这种更复杂的动力学情况，我们探索了适体结合的传统 4 态（“方形”）模型作为 K 的函数。将其扩展到这个附加维度会生成一个新的关于赖氨酸/K 结合的核糖开关折叠动力学“立方”模型，揭示核糖开关折叠（）和去折叠（）速率常数分别随 K 显著增加和减少。此外，温度依赖的单分子动力学研究表明，K 的存在在熵上增强了折叠的过渡态势垒，但通过增加赖氨酸结合的整体放热来部分补偿。我们将这种行为解释为证据，即 K 促进了赖氨酸的负电荷羧基与 RNA 之间的氢键形成，增加了结合口袋的结构刚性并降低了熵。最后，我们通过 Na 和 Cs 研究探索了阳离子大小的影响，以证明 K 最适合于赖氨酸和核糖开关适体结构域之间的桥接相互作用。因此，由核糖开关识别和结合赖氨酸的能力决定的赖氨酸产生和转运的调节与结合口袋中 K 的存在密切相关，并受到局部阳离子条件的强烈调节。结果表明，通过对细胞核糖开关环境中其他物质的敏感性，增加了赖氨酸核糖开关的功能。
