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：
7883479
负责人：
Thomas P Burghardt
金额：
$ 33.66万
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/7883479
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 Human Label Lead Learning Life 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 Rhodamines 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 public health relevance 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（由申请人提供）：肌球蛋白是一种分子运动结合ATP和肌动蛋白，可通过引起两种蛋白质的相对翻译来产生工作。肌球蛋白包含一个杠杆臂，可能是通过旋转〜70o的角度将肌动蛋白转换为电阻力来执行动力中风。肌球蛋白活性部位的ATP水解通过影响杠杆臂的运动通过肌动蛋白的肌动蛋白激活肌球蛋白ATPase的肌动蛋白的影响，通过影响杠杆臂的运动来供应收缩。影响是通过突变（自然发生和计算启发），分子动力学模拟（MD）和结构/功能测定法研究的耦合途径通过蛋白质基质进行的。确定用于研究的两种耦合途径介导肌球蛋白ATPase的肌动蛋白激活和构象变化触发色氨酸核苷酸敏感性，这可能将小型活性位点位移与较大的杠杆臂运动联系起来。该项目的目的是阐明肌动蛋白结合，活性位点构象，杠杆臂旋转和蛋白质位移之间的天然关系，然后观察这些关系如何受到引入耦合途径的修改的影响。人类骨骼肌球蛋白变体在运动生理，人类疾病和种群多样性中起着基本作用。这些变体涉及广泛分散的氨基酸取代，涵盖了功能所必需的几个区域，并且是通过耦合途径发现功能结构域相互联系的自然嵌入线索。它们暗示了模型蛋白中诱变的位点，对于肌球蛋白功能改变与表型是至关重要的。肌球蛋白MD模拟提供了耦合途径的互补见解。 MD介绍了在顺序时间内识别耦合网络的因果关系识别源，路径和终止，这是胜任电机的组成部分。适用于色氨酸核苷酸敏感性的因果关系测试已与补品平滑肌肌球蛋白的实验发现融合，以表明色氨酸核苷酸敏感性可能会与天然肌球蛋白的杠杆臂运动断开连接。一个新的实验性因果旋转/位移度量，量化生产性肌球蛋白周期的完成将使肌球蛋白杠杆臂旋转与体外测定中结合的肌动蛋白丝（F-肌动蛋白）的位移相关。两种分子技术利用绿色荧光蛋白（GFP）对肌球蛋白和纳米分辨率的分辨率定位与F-肌动蛋白结合的荧光探针。肌球蛋白变体适合于专业的功能，与人类疾病有关或在人群多样性中涉及的肌球蛋白变异，以深入了解功能差异。 MD模拟引入了因果关系，以表征肌球蛋白耦合网络并产生实验测试的假设。因果两分子测定测试完成生产性肌球蛋白周期的完成，并表征肌球蛋白的替代肌动蛋白的能力。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。公共卫生相关性：骨骼肌球蛋白是肌肉动力收缩的运动。它将化学能源转化为有用运动的能力对于我们过着幸福和生产力的生活的能力至关重要。拟议的研究促进了其对如何修复生病电动机的能源转化塑形方法的理解，以及如何使其适应肌肉生产力限制人类潜力的应用。