Synergism in the Molecular Crowding of Ligand-Induced Riboswitch Folding: Kinetic/Thermodynamic Insights from Single-Molecule Spectroscopy
Synergism in the Molecular Crowding of Ligand-Induced Riboswitch Folding: Kinetic/Thermodynamic Insights from Single-Molecule Spectroscopy
复制标题
配体诱导的核糖开关折叠的分子拥挤中的协同作用：来自单分子光谱的动力学/热力学见解
DOI：
10.1021/acs.jpcb.2c03507
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Nesbitt, David J.
中科院分区：
文献类型：
--
作者：
Sung, Hsuan-Lei;Nesbitt, David J.
核糖开关中的构象动力学涉及到RNA的配体结合和折叠，在活体细胞条件下，这两个过程都会受到排他体积效应的影响，因此在稀缓冲液条件下的体外研究中，这两个过程都是不完全的。本工作用温度相关的单分子荧光共振能量转移(FRET)光谱研究了(I)同系物配体和(II)分子簇合物(聚乙二醇、聚乙二醇单分子)折叠的热力学。枯草杆菌裂解半胱氨酸核糖开关。在详细的动力学分析的帮助下，我们分离和研究了聚乙二醇对赖氨酸结合步骤和核糖开关折叠步骤的影响，从中我们发现，聚乙二醇的拥挤主要通过增加与同源配体的亲和力来促进核糖开关折叠。依赖温度的研究进一步证实了这一点，研究表明，聚乙二醇的拥挤不是纯粹的熵，而是显著影响折叠的自由能景观的焓和熵贡献。结果表明，赖氨酸核糖开关的聚乙二醇单分子拥挤/稳定化在力学上更为复杂，并且需要超越传统的因排除体积和熵而产生的纯排斥的溶剂-溶质空间相互作用的图景。相反，目前的实验FRET数据支持另一种多步机制，即聚乙二醇首先以熵的方式将未折叠的核糖开关挤入“预折叠”构象，这反过来又极大地增加了配体结合亲和力，从而增强了核糖开关折叠的整体平衡。
Conformational dynamics in riboswitches involves ligand binding and folding of RNA, each of which can be influenced by excluded volume effects under “crowded”in vivocellular conditions and thus incompletely characterized byin vitrostudies under dilute buffer conditions. In this work, temperature-dependent single-molecule fluorescence resonance energy transfer (FRET) spectroscopy is used to characterize the thermodynamics of (i) cognate ligand and (ii) molecular crowders (PEG, polyethylene glycol) on folding of theB. subtilisLysClysine riboswitch. With the help of detailed kinetic analysis, we isolate and study the effects of PEG on lysine binding and riboswitch folding steps individually, from which we find that PEG crowding facilitates riboswitch folding primarily via a surprisingincrease in affinity for the cognate ligand. This is furthermore confirmed by temperature-dependent studies, which reveal that PEG crowding is not purely entropic and instead significantly impacts both enthalpic and entropic contributions to the free energy landscape for folding. The results indicate that PEG molecular crowding/stabilization of the lysine riboswitch is more mechanistically complex and requires extension beyond the conventional picture of purely repulsive solvent–solute steric interactions arising from excluded volume and entropy. Instead, the current experimental FRET data support an alternative multistep mechanism, whereby PEG first entropically crowds the unfolded riboswitch into a “pre-folded” conformation, which in turn greatly increases the ligand binding affinity and thereby enhances the overall equilibrium for riboswitch folding.