Single-molecule dynamics in solution with anti-Brownian trapping

在过去一年里，我们在以下两个方面取得了进展 A)在稀相中，类蛋白核蛋白形成复合体。最近，液-液相分离被发现驱动许多没有膜的细胞室(也被称为生物分子凝聚体)的组装，并成为细胞生物学中一个新兴的新范式。虽然大多数生物分子相分离的研究都集中在凝聚相上，但对稀相的研究相对较少。理论认为，在双组分相分离体系的稀相中形成稳定的络合物，影响相分离；然而，这些络合物还没有被实验所询问。我们证明了这样的复合体确实存在，使用了一个由纯化的蛋白Rubisco和EPYC1组成的相分离的细胞器--藻类蛋白核的体外重建系统。应用荧光相关光谱(FCS)测量扩散系数，我们发现在有或没有凝聚体存在的稀相中形成了络合物。这些络合物中的大多数恰好含有一个Rubisco分子。此外，我们开发了一个简单的分析模型，该模型概括了实验结果，并提供了对稀相组织的分子见解。因此，我们的结果证明了稀相中蛋白质复合体的存在，这可能在凝聚体的稳定性、动力学和调节中发挥重要作用。 B)Cas9全酶组装过程中gRNA构象的单分子解剖。生物分子通过循环通过一系列功能状态来实现其功能。为了更好地理解结构-功能关系，在功能状态的连续阶段探索结构是非常有意义的。我们最近使用Abel-FRET平台在单分子水平上探索了CRISPR RNA(CrRNA)的三端结构，因为它组装成Cas9全酶。对于每个分子，它的组装状态都是通过流体动力学轮廓明确地确定的，它的三端结构是通过一对战略放置的FRET染料来探测的。值得注意的是，我们在组装途径的每个阶段都发现了结构的异质性和动力学，即crRNA、引导RNA(gRNA或crRNA-trrRNA杂交)、Cas9-gRNA复合体和与底物DNA结合的Cas9-gRNA，突出了RNA结构多样性的重要性。目前的工作集中在使用RNA结构预测工具来生成与单分子FRET测量相一致的结构模型，并设计Cas9全酶组装的合理途径。这项工作可能有助于阐明Cas9-gRNA识别的基本生物物理原理 C)从单分子角度研究SARS-COV-2主要蛋白水解酶活性。SARS-COV-2的主要蛋白水解酶(MPRO)是冠状病毒复制和繁殖所必需的。MPRO以同源二聚体的形式存在，并负责前体多蛋白中的大多数成熟切割事件。由于其重要作用，MPRO一直是一个突出的药物靶点。例如，辉瑞公司的帕昔洛韦(PF-7321332)是一种Mpro的活性位点抑制剂。为了更好地了解MPRO的酶特性，并提供该药物如何扰乱蛋白酶的酶循环的机械性见解，我们的目标是开发一种新的单分子方法来观察单个MPRO酶对其底物的处理，一次一个分子。在这个项目的前6个月，我们已经建立了一个用于单分子观察的荧光标记MPRO蛋白的方案。我们还成功地在单分子水平上监测了二聚体-单体的平衡，验证了标记酶的活性。这些进展为未来MPRO的单分子酶学研究奠定了基础。

We have made progress on the following two areas during the past year a) phase-separating pyrenoid proteins form complexes in the dilute phase. Recently, liquid-liquid phase separation was found to drive the assembly of many cellular compartments that lack membranes (also referred to as biomolecular condensates) and became an emergent new paradigm in cellular biology. While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a simple analytical model which recapitulates experimental findings and provides molecular insights into the dilute phase organization. Thus, our results demonstrate the existence of protein complexes in the dilute phase, which could play important roles in the stability, dynamics, and regulation of condensates. b) Single-molecule dissection of gRNA conformation during Cas9 holoenzyme assembly. Biomolecules carry out their function by cycling through a series of functional states. To better understand the structural-functional relations, it is of tremendous interest to probe structure at sequential stages of the functional states. We recently used the ABEL-FRET platform to probe the 3-end structure of CRISPR RNA (crRNA) at the single-molecule level as it assembles into the Cas9 holoenzyme. For every molecule, its assembly state is unambiguously determined using hydrodynamic profiling and its 3-end structure is probed by a pair of strategically placed FRET dyes. Strikingly, we discovered structural heterogeneity and dynamics at every stage of the assembly pathway that is, crRNA, guide RNA (gRNA, or crRNA-tracrRNA hybrid), Cas9-gRNA complex and Cas9-gRNA bound with substrate DNA, highlighting the importance of RNA structural diversity. Current work focuses on using RNA structural prediction tools to generate structural models consistent with single-molecule FRET measurements and devising plausible pathways of Cas9 holoenzyme assembly. This work could potentially shed light on fundamental biophysical principles of Cas9-gRNA recognition c) Towards a single-molecule view of SARS-COV-2 main protease activity. The main protease of SARS-COV-2 (MPro) is indispensable for the coronavirus replication and propagation. MPro exists as a homodimer and is responsible for most maturation cleavage events within the precursor polyprotein. Due to its vital roles, MPro has been a prominent drug target. For example, Paxlovid (PF-7321332) from Pfizer is an active site inhibitor of MPro. To better understand the enzymatic properties of MPro and provide mechanistic insights of how the drug disrupts the enzymatic cycle of the protease, we aim to develop a new single-molecule assay to watch individual MPro enzymes process their substrates, one molecule at a time. In the first 6 months of this project, we have established a protocol to fluorescently label Mpro proteins for single-molecule observations. We have also successfully monitored the dimer-monomer equilibrium at the single-molecule level which validated the activity of the labeled enzyme. These progress set the stage to future single-molecule enzymology studies of MPro.