In Situ Sensing of Single Myosin Function in Hypertrophy Disease

肥厚性疾病中单一肌球蛋白功能的原位传感

基本信息

批准号：
7981390
负责人：
Thomas P Burghardt
金额：
$ 37.77万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-15 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7981390
关键词：
3-Dimensional ATP Hydrolysis Actins Active Sites Affect Affinity Binding Binding Sites Biological Assay Calcium Cardiac Cardiac Myosins Cardiomegaly Complex Crowding Crystallization Data Detection Disease Docking Effectiveness Electrons Environment Familial Hypertrophic Cardiomyopathy Fluorescence Polarization Free Energy Gene Mutation Genetic Screening Goals H-Meromyosin Heart Hypertrophy Image Imagery In Situ In Vitro Individual Kinetics Label Light Link Location Measures Mechanics Mediating Medicine Metric Microscopic Modeling Molecular Molecular Conformation Molecular Motors Monitor Motor Movement Muscle Muscle Fibers Muscle Rigidity Muscle function Mutate Mutation Myosin ATPase Myosin Heavy Chains Myosin Regulatory Light Chains Myosin Type II Pathway interactions Peptides Persons Phosphorylation Play Positioning Attribute Power stroke Production Proteins Recombinants Regulation Relative (related person)Research Role Rotation Single Nucleotide Polymorphism Site Smooth Muscle Structure System Testing Time Tissues Torque Translating Translations Tryptophan Work arm base cell motility dipole moment disease phenotype inorganic phosphate mutant papillary muscle protein function protein structure function public health relevance reconstruction research study single molecule src Homology Region 2 Domain sudden cardiac death

项目摘要

DESCRIPTION (provided by applicant): Genetic screening has detected abundant mutations in sarcomeric proteins elucidating basic causes for disease and identifying targets for individualized medicine when a functional deficit on the protein level can be identified. The project focuses on the molecular motor myosin and its regulation using various approaches for the expression, dynamical characterization, and structural visualization of the protein in its native and mutated forms. The goal is to decipher the role individual mutations play in modifying native myosin function. Myosin performs ATP free energy transduction into mechanical work by coordinating ATP hydrolysis at the active site, actin affinity modulation at the actin binding site, and the lever-arm power stroke, via allosteric transduction pathways operating in a time ordered sequence. Energy transduction is the definitive systemic feature of myosin and a working model for native transduction allocates specific functions to structural domains within the motor beginning with ATP hydrolysis in the active site and ending in a power stroke rotating a lever- arm domain in the motor through ~70 degrees in the crowded environment of the muscle tissue. The cardiac myosin heavy chain (MHC) and both of its light chains (MLCs) harbor familial hypertrophic cardiomyopathy (FHC)-linked mutations. MHC mutants are hypothesized to disrupt specific transduction pathways. Evolutionarily conserved allosteric connectivity prediction identifies residues in MHC forming the transduction pathway. Transduction pathway residues that are also FHC-linked mutation sites identify the MHC candidate mutants affecting transduction. Several MLC mutants are hypothesized to impact lever-arm structural stability influencing lever-arm dynamics and effectiveness. Myosin modified by a disease-linked MHC or MLC candidate mutation is subjected to in vitro and in situ experiments to determine how the mutations impact, the functional domains in MHC operating in a working model for native transduction, or the lever-arm stability provided by the MLC. A single molecule experiment detecting lever-arm rotary movement is especially pertinent because it is applicable to myosin in the native crowded environment of the muscle fiber. Myosin regulatory light chain (RLC) may have special significance because it is partially phosphorylated at Ser15 in normal cardiac tissue. Phosphorylation apparently affects myosin calcium regulation while in the muscle tissue and myosin duty ratio in vitro within single myosin motors. In the latter case, RLC conformation modulation by phosphorylation must impact myosin function related to strong actin binding. RLC crystallization and structure determination will investigate the structural basis of RLC regulation of myosin as well as the impact of FHC-linked mutations on RLC structure. PUBLIC HEALTH RELEVANCE: Familial hypertrophic cardiomyopathy (FHC) is a disease characterized by an enlarged heart. It affects 1 in 500 persons and is a cause of sudden cardiac death in the young. Genetic mutations affecting protein structure and function in the heart are linked to FHC. The project goal is to associate the mutation with the specific protein function affected to identify basic causes for disease and targets for individualized medicine. The proposed research applies promising new computational and experimental approaches for assessing how disease implicated mutations change the motor powering contraction.

描述（由申请人提供）：遗传筛查已检测到透明的肌肉蛋白中的大量突变，阐明了疾病的基本原因，并确定可以确定蛋白质水平的功能不足时的个性化医学靶标。该项目着重于分子运动肌球蛋白及其调节，采用各种方法来表达，动力学表征和蛋白质以其天然和突变形式的结构可视化。目的是破译单个突变在修改天然肌球蛋白功能中所扮演的角色。肌球蛋白通过在活性位点协调ATP水解，肌动蛋白结合位点的肌动蛋白亲和力调节以及通过在时间顺序的序列上运行的变构传输途径来执行ATP自由能将其转导向机械工作。能量转导是肌球蛋白的确切系统特征，而用于天然转导的工作模型则是在电动部位的ATP水解开始，将特定功能分配给电动机内的结构域，并以动力冲程在肌肉组织拥挤环境中的〜70度旋转的电动杆域中旋转杆杆域。心脏肌球蛋白重链（MHC）及其两个光链（MLC）都具有家族性肥厚性心肌病（FHC）连接突变。假设MHC突变体破坏特定的转导途径。进化保守的变构连通性预测确定了形成转导途径的MHC中的残基。也是FHC连接突变位点的转导途径残基鉴定了影响转导的MHC候选突变体。假设几个MLC突变体会影响杠杆臂结构稳定性，从而影响杠杆动力和有效性。通过疾病连接的MHC或MLC候选突变修饰的肌球蛋白受到体外和原位实验的进行，以确定突变的影响，在工作模型中运行的MHC中的功能域或MLC提供的杠杆臂稳定性。一个单个分子实验检测杠杆臂旋转运动是特别相关的，因为它适用于肌肉纤维的天然拥挤环境中的肌球蛋白。肌球蛋白调节轻链（RLC）可能具有特殊的意义，因为它在正常心脏组织中部分磷酸化。磷酸化显然会影响肌球蛋白钙调节，而在单个肌球蛋白电动机内的肌肉组织和肌球蛋白占用比。在后一种情况下，磷酸化的RLC构象调节必须影响与强肌动蛋白结合有关的肌球蛋白功能。 RLC的结晶和结构确定将研究肌球蛋白的RLC调节的结构基础，以及FHC连接突变对RLC结构的影响。公共卫生相关性：家族性肥厚性心肌病（FHC）是一种以肿大心脏为特征的疾病。它影响了500人中有1人，是年轻人突然心脏死亡的原因。影响蛋白质结构和心脏功能的基因突变与FHC有关。项目目标是将突变与特定蛋白质功能相关联，以确定疾病的基本原因和个性化医学靶标。拟议的研究采用了有希望的新计算方法和实验方法来评估疾病涉及的突变如何改变运动动力收缩。