Role of electric fields and a dynamic local environment in protein-RNA molecular recognition

核糖核酸(RNA)是一种具有大量负电荷的生物聚合物，与它们结合的蛋白质表面通常含有带电荷的氨基酸残基，这有助于吸引、指导和结合它们的靶标。重要的是，这些都是大而灵活的分子，所以它们的结构和局部环境的波动对它们的功能起着重要的作用。这个项目试图通过开发一种结合了各种光谱技术的多模式仪器来了解RNA和蛋白质之间的分子识别过程是如何受到它们的静电相互作用和发生结合的动态环境的影响的。这项研究将培训学生在物理化学和生物物理学的界面上掌握多学科技能，产生更广泛的影响，包括开发可转移的方法来定量表征生物分子相互作用。在这个项目中，用于远程学习的可扩展和定量的实验套件将被设计和实施，目标是将动手、基于经验的教育扩展到典型的教学实验室环境之外。蛋白质和RNA之间的相互作用显示出显著的多样性-蛋白质可以识别其RNA靶标的方式特定于其序列、结构、两者或两者都不特定。为了揭示调节底物招募、复杂稳定性和多个蛋白质结构域协作目标识别的分子尺度机制，该项目整合了电化学和光谱工具，以测量蛋白质-RNA结合的动态特征、它们的构象波动以及它们对非平衡扰动的反应--所有这些都是在受控静电环境的影响下进行的。RNA底物将被定位在电极表面，以调整其与溶液中蛋白质的静电相互作用，所产生的蛋白质-RNA复合体将使用结合电化学与中红外等离子体、超快多通道荧光和泵浦-探针光谱的多模式仪器进行探测。这项研究的结果将揭示静电相互作用和生物分子结构和溶剂化的快速动力学如何影响蛋白质-RNA复合体的形成、稳定性和解离的热力学和动力学。这个项目得到了分子和细胞生物科学部分子生物物理学分部的支持。这个奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Ribonucleic acids (RNA) are biopolymers with substantial amounts of negative electrical charge, and the surfaces of proteins that bind them often contain amino acid residues with electrical charge that can help attract, direct, and bind their targets. Importantly, these are large and flexible molecules so fluctuations in their structure and local environment play a significant role in their function. This project seeks to understand how the process of molecular recognition between RNA and proteins is influenced by their electrostatic interactions and by the dynamic environment in which binding occurs by developing a multimodal instrumentation that combines a variety of spectroscopic techniques. This research will train students in a multidisciplinary skillset at the interface of physical chemistry and biophysics, with broader impacts that include the development of transferrable methods for quantitative characterization of biomolecular interactions. In this project, scalable and quantitative experiment kits for remote learning will be designed and implemented with the goal of expanding hands-on, experience-based education beyond typical teaching laboratory settings.Interactions between proteins and RNA show significant diversity – proteins can recognize their RNA targets in ways that are specific to their sequence, to their structure, to both, or to neither. To uncover the molecular-scale mechanisms that tune substrate recruitment, complex stability, and cooperative target recognition by multiple protein domains, this project integrates electrochemical and spectroscopic tools to measure the dynamic signatures of protein-RNA binding, their conformational fluctuations, and their response to non-equilibrium perturbations – all under the effects of a controlled electrostatic environment. RNA substrates will be localized at an electrode surface to tune their electrostatic interactions with proteins in solution, and the resulting protein-RNA complexes will be probed with a multimodal instrument that combines electrochemistry with mid-infrared plasmonics, ultrafast multi-channel fluorescence, and pump-probe spectroscopies. The outcomes of this research will reveal the way electrostatic interactions and fast dynamics of biomolecular structure and solvation affect the thermodynamics and kinetics of formation, stability, and dissociation of protein-RNA complexes. This project is supported by the Molecular Biophysics Cluster of the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.