权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.

项目总结