Theoretical and experimental investigation of multi-domain protein folding and conformational dynamics

多域蛋白质折叠和构象动力学的理论和实验研究

基本信息

批准号：
10218203
负责人：
JIN WANG
金额：
$ 45.81万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10218203
关键词：
Address Binding Biological Process Biology Bypass Catalysis Cell physiology Cells Chemicals Circular Dichroism Closure by clamp Collaborations Crowding DNA DNA Binding DNA Damage DNA lesion DNA-Directed DNA Polymerase Elements Energy Transfer Environment Family Fluorescence Fluorescence Spectroscopy Hot Spot Human In Vitro Investigation Ions Kinetics Laboratories Laboratory Research Lobe Measurement Methods Microscopic Modeling Molecular Molecular Conformation Names Nucleotides Organism Pathway interactions Play Polymerase Principal Investigator Process Proliferating Cell Nuclear Antigen Protein Conformation Protein Dynamics Protein Engineering Proteins Publishing Research Personnel Ribosomes Role Slide Structure Sulfolobus solfataricus System Tertiary Protein Structure Testing Theoretical Studies Theoretical model Uncertainty Validation Work antigen binding base conformational conversion drug discovery experimental study flexibility improved in vivo macromolecule novel predictive modeling protein folding replication factor A stopped-flow fluorescence thermostability three dimensional structure

项目摘要

PROJECT SUMMARY Proper folding is crucial to achieving a protein’s unique three dimensional structure while the conformational dynamics of the protein play a major role in its biological function. Although significant progress has been made in understanding the folding/unfolding and conformational dynamics for single-domain proteins, these two fundamental processes remain largely unexplored for multi-domain proteins, which have been suggested to account for up to 80% of all eukaryotic proteins. Therefore, a significant disparity exists in our understanding of the underlying mechanisms of folding/unfolding and conformational dynamics for the majority of human proteins and we seek to address these uncertainties through theoretical and experimental investigation. In this proposal, we devise a comprehensive strategy to answer the above, in-depth mechanistic unknowns regarding multi-domain proteins through an energy landscape approach with subsequent experimental validation. The energy landscape approach significantly improves technical capabilities through the establishment of theoretical models for uncovering underlying mechanisms. By establishing the microscopic energy landscape and structure based models, we will elucidate the folding/unfolding mechanisms of DPO4, a multi-domain, model Y-family DNA polymerase critical for bypassing unrepaired DNA lesions, in vitro and in vivo (here means mimicking in vivo conditions), and predict possible intermediate states and critical residues under various environments, including the presence of the ribosome (co-translational) and a crowding agent (in vivo), as well as different thermal and chemical denaturant conditions. Through our microscopic energy landscape and structure based models, we will reveal the underlying mechanisms of conformational changes between various conformational states of DPO4 upon binding to DNA or a protein replication factor PCNA through quantifying the stability, kinetics, and structural hot spots critical for function. The theoretical model predictions will be tested and validated through stopped-flow, circular dichroism, fluorescence energy transfer, and other spectroscopic experiments. The results generated from the proposal will advance the DNA polymerase field while the methods developed here are general and can serve as a framework for studies of folding/unfolding and conformational dynamics of other multi-domain proteins. Moreover, the intricacies of protein folding/unfolding and conformational transitions revealed by our proposed studies will facilitate protein design and drug discovery.

项目概要正确的折叠对于实现蛋白质独特的三维结构至关重要，而构象尽管已经取得了重大进展，但蛋白质的动力学在其生物学功能中发挥着重要作用。为了理解单域蛋白质的折叠/展开和构象动力学，这些对于多结构域蛋白质，两个基本过程在很大程度上仍未被探索，这已被提出占所有真核蛋白质的 80% 因此，我们的理解存在显着差异。大多数人类折叠/展开和构象动力学的基本机制蛋白质，我们试图通过理论和实验研究来解决这些不确定性。提案中，我们设计了一个全面的策略来回答上述关于机械的深入的未知问题通过能量景观方法和随后的实验验证来研究多域蛋白质。能源景观方法通过建立通过建立微观能量景观来揭示潜在机制的理论模型。和基于结构的模型，我们将阐明 DPO4（一种多域、模型 Y 家族 DNA 聚合酶对于绕过体外和体内未修复的 DNA 损伤至关重要（这里是指模仿体内条件），并预测各种情况下可能的中间状态和关键残基环境，包括核糖体（共翻译）和拥挤剂（体内）的存在，以及通过我们的微观能量景观和不同的热和化学变性条件。基于结构的模型，我们将揭示不同结构之间构象变化的潜在机制通过定量分析 DPO4 与 DNA 或蛋白质复制因子 PCNA 结合后的构象状态对功能至关重要的稳定性、动力学和结构热点。通过停流、圆二色性、荧光能量转移等进行测试和验证该提案产生的结果将推动 DNA 聚合酶领域的发展。虽然这里开发的方法是通用的，可以作为折叠/展开研究的框架以及其他多结构域蛋白质的构象动力学。我们提出的研究揭示的折叠/展开和构象转变将促进蛋白质设计和药物发现。