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Visualizing Actomyosin Transients by Data Merging

通过数据合并可视化肌动球蛋白瞬变

基本信息

批准号：
8288322
负责人：
Thomas P Burghardt
金额：
$ 32.31万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-04-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8288322
关键词：
ATP Hydrolysis ATP phosphohydrolase ATP-G-actin Abbreviations Actins Active Sites Actomyosin Address Affect Amino Acid Substitution Amino Acids Aorta Area Binding Binding Sites Biological Assay Cardiac Chemicals Chickens Circular Dichroism Coupled Coupling Data Dictyostelium discoideum Disease Etiology Exercise Physiology F-Actin Fluorescence Fluorescence Microscopy Fluorescent Probes Free Energy Freedom Goals Green Fluorescent Proteins H-Meromyosin Head Health Human Label Lead Learning Light Link Lysine Mediating Methods Metric Microfilaments Microscopy Mining Modeling Modification Molecular Conformation Molecular Motors Motor Movement Muscle Mutagenesis Mutation Myopathy Myosin ATPase Myosin Heavy Chains Myosin Light Chain Kinase Myosin Light Chains Myosin Regulatory Light Chains Myosin S-1 Nucleotides Occupations Pathology Pathway interactions Peptides Phenotype Play Population Positioning Attribute Power stroke Probability Productivity Protein Isoforms Proteins Recombinant Proteins Relative (related person)Research Resolution Rhodamine Role Rotation Shapes Site Smooth Muscle Myosins Sorting - Cell Movement Source Structure Surface System Techniques Testing Thermodynamics Time Translating Translations Tryptophan Variant Work acronyms analytical tool arm base cell motility density design human disease in vitro Assay insight molecular dynamics mutant nanometer next generation repaired sensor simulation skeletal transmission process

项目摘要

DESCRIPTION (provided by applicant): Myosin is a molecular motor binding ATP and actin to produce work by causing relative translation of the two proteins. Myosin contains a lever arm probably executing a power stroke by rotating through an angle of ~70o to translate actin against resistive force. ATP hydrolysis at myosin's active site energizes contraction by influencing lever arm movement and is influenced by allostery with actin in actin-activation of myosin ATPase. The influences are conducted through the protein matrix by coupling pathways investigated by mutation (naturally occurring and computation inspired), molecular dynamics simulation (MD), and structure/function assays. Two coupling pathways identified for study mediate actin-activation of myosin ATPase and conformation change triggering tryptophan nucleotide sensitivity that might link small active site displacements to the larger lever arm movement. The goal of the project is to elucidate the native relationships among actin binding, active site conformation, lever arm rotation and protein displacement and then to observe how these relationships are affected by modifications introduced to coupling pathways. Human skeletal myosin variants play a fundamental role in exercise physiology, human disease, and population diversity. The variants involve widely dispersed amino acid substitutions covering several regions essential to function and are naturally embedded clues to discovering functional domain interconnectedness through the coupling pathways. They implicate sites for mutagenesis in model proteins and are essential for correlation of myosin functional alteration to phenotype. Myosin MD simulation provides complementary insights into how coupling pathways perform. MD introduces the causality test identifying source, path, and termination of coupling networks in sequential time that is an integral part of the competent motor. Causality testing applied to tryptophan nucleotide sensitivity has converged with experimental findings from a tonic smooth muscle myosin to suggest tryptophan nucleotide sensitivity could disconnect from lever arm movement in native myosin. A new experimental causal rotation/displacement metric, quantifying completion of a productive myosin cycle, will correlate myosin lever arm rotation with displacement of a bound actin filament (F-actin) in an in vitro assay. The two-molecule technique utilizes a green fluorescent protein (GFP) on myosin and nanometer resolution localization of a fluorescent probe bound to F-actin. Myosin variants that are, adapted to specialize function, implicated in human disease, or sourced in population diversity, are mined for insight into functional divergence. MD simulation introduces causality to characterize myosin coupling networks and produces experimentally testable hypotheses. A causal two-molecule assay tests completion of a productive myosin cycle and characterizes myosin's ability to displace actin. These analytical tools are next-generation methods addressing transduction and motility in muscle myosin. PUBLIC HEALTH RELEVANCE: Skeletal myosin is the motor in muscle powering contraction. Its ability to convert chemical energy to useful movement is fundamental to our ability to lead happy and productive lives. The proposed research promotes understanding of its design for energy conversion shaping approaches for how to repair an ailing motor and how to adapt it to applications where muscle productivity is limiting human potential.

描述(申请人提供)：肌球蛋白是一种结合ATP和肌动蛋白的分子马达，通过导致这两种蛋白质的相对翻译来产生功。肌球蛋白含有一个杠杆臂，可能通过旋转约70度的角度来执行力量打击，以翻译肌动蛋白对抗阻力。肌球蛋白活性部位的ATP水解物通过影响杠杆臂的运动使收缩充满能量，并在肌球蛋白ATPase的肌动蛋白激活过程中受肌动蛋白变构的影响。通过突变(自然发生和计算启发)、分子动力学模拟(MD)和结构/功能分析研究的耦合通路，这些影响通过蛋白质基质进行。研究中发现的两条偶联通路介导了肌球蛋白ATPase的肌动蛋白激活和构象变化，触发了色氨酸核苷酸敏感性，可能将小的活性部位移位与较大的杠杆臂运动联系起来。该项目的目标是阐明肌动蛋白结合、活性部位构象、杠杆臂旋转和蛋白质位移之间的天然关系，然后观察偶联途径的修饰如何影响这些关系。人类骨骼肌球蛋白变异在运动生理学、人类疾病和种群多样性中发挥着重要作用。这些变体涉及广泛分布的氨基酸替换，覆盖了几个功能必需的区域，自然地嵌入了通过偶联途径发现功能结构域互连的线索。它们暗示了模型蛋白中突变的位置，并且对于肌球蛋白功能改变与表型的关联是必不可少的。肌球蛋白MD模拟提供了对偶联通路如何执行的补充见解。MD介绍了在顺序时间内识别耦合网络的来源、路径和终端的因果检验，这是合格电机的组成部分。应用于色氨酸核苷酸敏感性的因果关系测试与紧张性肌球蛋白的实验结果一致，表明在天然肌球蛋白中，色氨酸核苷酸敏感性可能与杠杆手臂运动脱节。一种新的实验性因果旋转/位移度量，量化了一个生产性肌球蛋白周期的完成，将在体外实验中将肌球蛋白杠杆臂旋转与结合的肌动蛋白细丝(F-肌动蛋白)的移位相关联。双分子技术利用肌球蛋白上的绿色荧光蛋白(GFP)和与F-肌动蛋白结合的荧光探针的纳米分辨率定位。挖掘肌球蛋白变异是为了深入了解功能差异，这些变异适合于专门化功能，与人类疾病有关，或源于种群多样性。MD模拟引入了因果关系来描述肌球蛋白偶联网络，并产生了实验上可测试的假说。一个因果双分子分析测试了一个有效的肌球蛋白循环的完成，并表征了肌球蛋白取代肌动蛋白的能力。这些分析工具是解决肌肉肌球蛋白转导和运动的下一代方法。与公共健康相关：骨骼肌球蛋白是肌肉动力收缩的马达。它将化学能转化为有用的运动的能力是我们过上幸福和富有成效的生活的基础。这项拟议的研究促进了人们对其能量转换设计、如何修复故障马达以及如何使其适应肌肉生产力限制人类潜力的应用的理解。