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Mechanochemistry of Myosin II Filaments

肌球蛋白 II 丝的机械化学

基本信息

批准号：
10203824
负责人：
Jonathan E. Baker
金额：
$ 40.94万
依托单位：
UNIVERSITY OF NEVADA RENO
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-07 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10203824
关键词：
ATP phosphohydrolase Actins Address Affect Binding Biological Assay Biological Models Calcium Cardiac Cardiac Myosins Chemicals Chemistry Computer software Data Data Set Dependence Disease Drosophila genus Equation Experimental Designs Filament Genetic Engineering Geometry Goals Head Heart In Vitro Insecta Kinetics Label Laboratories Length Literature Measurement Measures Mechanics Methods Microfilaments Modeling Molecular Motion Movement Muscle Muscle Contraction Myocardium Myopathy Myosin ATPase Myosin Heavy Chains Myosin Regulatory Light Chains Myosin Type II Organ Patients Pharmaceutical Preparations Phosphorylation Physiological Post-Translational Protein Processing Process Protein Dephosphorylation Publishing Quantum Dots Recombinants Regulation Reporting Sarcomeres Skeletal Muscle Slide Smooth Muscle Smooth Muscle Myosins Structure System Techniques Testing Thick Filament Thin Filament Time Work cell motility data standards experimental study in vitro Model mechanical load monomer novel reconstitution single molecule skeletal

项目摘要

Project Summary This project will explore the mechanochemistry of myosin II filaments moving on actin filaments using in vitro methods. We will use our recently-developed inverted motility assay to measure myosin filament velocities moving on actin filaments. By varying the number of myosin heads in the filaments and the ATP concentration, we have developed a deterministic model describing the parameters that define and limit the rate of filament- filament sliding that inform mechanisms of muscle contraction. Our prior work revealed how assembling myosin into filaments allows for attachment-limited kinetics at physiological numbers of myosins in the filaments, in contrast to the current paradigm in the field of detachment-limited kinetics. We use this assay as the basis for addressing further specific structural and kinetic hypotheses about how force and motion are generated when myosin is incorporated into a filament. Myosin filaments, both reconstituted and native, from smooth, skeletal, insect flight, and cardiac muscle will be compared. Genetically engineered fruit flies will be generated to express insect flight muscle myosin with either shortened or lengthened S2 domains, which is the myosin heavy chain domain that we hypothesize to underlie the attachment-limited kinetics mentioned above. The effects of phosphorylation of the myosin regulatory light chains will be examined, with the goal of testing a novel hypothesis about the activity of myosin with only one of its heads phosphorylated. The assay will be modified in two ways to extend its utility. First, single myosin heads will be labeled with quantum dots an incorporated into co-filaments. The global motion of the moving filament and the quantum dot within the moving filament will simultaneously visualized, and by analysis of the motion by tracking software, the presence or absence of predicted mechanical signatures will be assessed. The identity of those signatures will be assessed by correlation to changing experimental conditions that we predict will change the signature. Also, the inverted in vitro motility assay will be modified to allow measurement of myosin filament moving under load, allowing underlying effects of load on kinetics to be examined and allow comparisons of the relative ability of different myosins to generate power. Final, we will build on these approaches by using the inverted motility assay to probe mechanisms underlying the regulation of thin filament –thick filament sliding by calcium and myosin head binding.

项目总结

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The myosin duty ratio tunes the calcium sensitivity and cooperative activation of the thin filament.

肌球蛋白占空比调节细丝的钙敏感性和协同激活。

DOI：
10.1021/bi400262h
发表时间：
2013
期刊：
Biochemistry
影响因子：
2.9
作者：
Webb,Milad;JacksonJr,DelR;Stewart,TravisJ;Dugan,SamuelP;Carter,MichaelS;Cremo,ChristineR;Baker,JoshE
通讯作者：
Baker,JoshE

Synthesis and Evaluation of 4-Hydroxycoumarin Imines as Inhibitors of Class II Myosins.

DOI：
10.1021/acs.jmedchem.0c01062
发表时间：
2020-10-08
期刊：
Journal of medicinal chemistry
影响因子：
7.3
作者：
Brawley J;Etter E;Heredia D;Intasiri A;Nennecker K;Smith J;Welcome BM;Brizendine RK;Gould TW;Bell TW;Cremo C
通讯作者：
Cremo C

A mixed-kinetic model describes unloaded velocities of smooth, skeletal, and cardiac muscle myosin filaments in vitro.