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Single-molecule dynamics in solution with anti-Brownian trapping

具有反布朗捕获的溶液中的单分子动力学

基本信息

批准号：
10919534
负责人：
Quan Wang
金额：
$ 121.72万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10919534
关键词：
2019-nCoV Active Sites Area Binding Biological Assay Biophysics Cellular biology Clustered Regularly Interspaced Short Palindromic Repeats Complex Coronavirus DNA Development Diffusion Dissection Drug Targeting Dyes Enzymatic Biochemistry Enzymes Equilibrium Event Fluorescence Fluorescence Resonance Energy Transfer Future Goals Guide RNA Heterogeneity Holoenzymes Hybrids In Vitro Individual Label Liquid substance Measurement Measures Membrane Modality Modeling Molecular Molecular Conformation Monitor Organelles Pathway interactions Paxlovid Peptide Hydrolases Pharmaceutical Preparations Phase Physical condensation Play Polyproteins Process Property Proteins Protocols documentation RNA RNA Conformation Reaction Regulation Ribulose-Bisphosphate Carboxylase Role SARS-CoV-2 protease Series Spectrum Analysis Structural Models Structure System Time Work dimer inhibitor insight interest monomer novel predictive tools protein complex protein purification reconstitution single molecule single-molecule FRET theories tool

项目摘要

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.

过去一年，我们在以下两方面取得了进展 a）相分离蛋白核蛋白在稀相中形成复合物。最近，发现液-液相分离驱动许多缺乏膜的细胞区室（也称为生物分子缩合物）的组装，并成为细胞生物学中出现的新范例。虽然大多数生物分子相分离的研究都集中在凝聚相，相对较少的是关于稀相。理论表明，在双组分相分离系统的稀相中形成稳定的复合物，影响相分离;然而，这些复合物尚未被实验询问。我们表明，这样的复合物确实存在，使用体外重建系统的相分离的细胞器，藻蛋白核，由纯化的蛋白质Rubisco和EPYC 1。应用荧光相关光谱（FCS）测量扩散系数，我们发现，在稀相或不存在冷凝物的情况下，形成复合物。这些复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，概括了实验结果，并提供了稀相组织的分子见解。因此，我们的研究结果表明，蛋白质复合物在稀相中的存在，这可能发挥重要作用的稳定性，动力学，和调节的冷凝物。 B）在Cas9全酶组装期间gRNA构象的单分子解剖。生物分子通过一系列功能状态的循环来执行其功能。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趣的。我们最近使用ABEL-FRET平台在单分子水平上探测CRISPR RNA（crRNA）的3端结构，因为它组装成Cas9全酶。对于每一个分子，其组装状态是明确确定使用流体动力学分析和其3-端结构探测一对战略性放置的FRET染料。引人注目的是，我们在组装途径的每个阶段都发现了结构异质性和动力学，即crRNA，指导RNA（gRNA或crRNA-tracrRNA杂合体），Cas9-gRNA复合物和与底物DNA结合的Cas9-gRNA，突出了RNA结构多样性的重要性。目前的工作重点是使用RNA结构预测工具来生成与单分子FRET测量一致的结构模型，并设计Cas9全酶组装的合理途径。这项工作可能揭示Cas9-gRNA识别的基本生物物理原理 c）SARS-COV-2主要蛋白酶活性的单分子观点。SARS-COV-2的主要蛋白酶（MPro）是冠状病毒复制和繁殖所必需的。MPro作为同源二聚体存在，并且负责前体多蛋白内的大多数成熟裂解事件。由于其重要作用，MPro一直是一个重要的药物靶点。例如，来自Pfizer的Paxlovid（PF-7321332）是MPro的活性位点抑制剂。为了更好地了解MPro的酶特性，并提供药物如何破坏蛋白酶的酶循环的机制见解，我们的目标是开发一种新的单分子测定法，以观察单个MPro酶处理其底物，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协议，荧光标记Mpro蛋白的单分子观察。我们还成功地监测了单分子水平的二聚体-单体平衡，验证了标记酶的活性。这些进展为MPro的单分子酶学研究奠定了基础。