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：
8281567
负责人：
Thomas P Burghardt
金额：
$ 39.03万
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/8281567
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 function Mutate Mutation Myocardium 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）：基因筛查检测到肌节蛋白中的大量突变，阐明了疾病的基本原因，并在可以识别蛋白质水平的功能缺陷时确定了个体化医疗的目标。该项目重点研究分子运动肌球蛋白及其调节，使用各种方法对天然和突变形式的蛋白质进行表达、动态表征和结构可视化。目标是破译个体突变在改变天然肌球蛋白功能中所起的作用。肌球蛋白通过按时间顺序运行的变构转导途径协调活性位点的 ATP 水解、肌动蛋白结合位点的肌动蛋白亲和力调节以及杠杆臂动力冲程，将 ATP 自由能转导为机械功。能量转导是肌球蛋白的明确系统特征，天然转导的工作模型将特定功能分配给电机内的结构域，从活性位点的 ATP 水解开始，到动力冲程结束，使电机中的杠杆臂域旋转通过~ 70度处于肌肉组织拥挤的环境中。心肌肌球蛋白重链 (MHC) 及其两条轻链 (MLC) 含有家族性肥厚型心肌病 (FHC) 相关突变。假设 MHC 突变体会破坏特定的转导途径。进化保守的变构连接预测可识别 MHC 中形成转导途径的残基。转导途径残基也是 FHC 相关突变位点，可识别影响转导的 MHC 候选突变体。假设几种 MLC 突变体会影响杠杆臂结构稳定性，从而影响杠杆臂动力学和有效性。通过与疾病相关的 MHC 或 MLC 候选突变修饰的肌球蛋白进行体外和原位实验，以确定突变如何影响、在天然转导工作模型中运行的 MHC 功能域，或由MLC。检测杠杆臂旋转运动的单分子实验特别相关，因为它适用于肌纤维天然拥挤环境中的肌球蛋白。肌球蛋白调节轻链 (RLC) 可能具有特殊意义，因为它在正常心脏组织中 Ser15 部分被磷酸化。磷酸化明显影响肌肉组织中的肌球蛋白钙调节和体外单肌球蛋白运动中的肌球蛋白占空比。在后一种情况下，磷酸化引起的 RLC 构象调节必定会影响与强肌动蛋白结合相关的肌球蛋白功能。 RLC结晶和结构测定将研究RLC调节肌球蛋白的结构基础以及FHC相关突变对RLC结构的影响。公众健康相关性：家族性肥厚型心肌病 (FHC) 是一种以心脏扩大为特征的疾病。每 500 人中就有 1 人受到影响，是年轻人心源性猝死的一个原因。影响心脏蛋白质结构和功能的基因突变与 FHC 有关。该项目的目标是将突变与受影响的特定蛋白质功能联系起来，以确定疾病的基本原因和个体化医疗的目标。拟议的研究应用了有前景的新计算和实验方法来评估疾病相关突变如何改变运动动力收缩。