In Situ Sensing of Single Myosin Function in Hypertrophy Disease

描述(申请人提供)：基因筛查在肌瘤蛋白中检测到大量突变，阐明了疾病的基本原因，并在可以确定蛋白质水平上的功能缺陷时确定了个体化药物的靶点。该项目专注于分子马达肌球蛋白及其调控，使用各种方法来表达、动态表征和结构可视化天然和突变形式的蛋白质。其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。肌球蛋白通过时序变构转导途径协调活性部位的ATP水解、肌动蛋白结合部位的肌动蛋白亲和力调节以及杠杆-手臂功率卒中，从而将ATP自由能转化为机械功。能量转导是肌球蛋白明确的系统特征，天然转导的工作模型将特定的功能分配给运动内的结构域，从活动部位的ATP水解开始，以动力卒中结束，在肌肉组织拥挤的环境中，将运动中的杠杆-臂域旋转约70度。心肌肌球蛋白重链(MHC)及其轻链(MLCs)含有家族性肥厚型心肌病(FHC)相关突变。MHC突变体被认为会破坏特定的转导途径。进化保守的变构连接性预测确定了形成转导途径的MHC中的残基。也是FHC连锁突变位点的转导途径残基识别影响转导的MHC候选突变体。几个MLC突变体被假设影响杠杆-手臂结构的稳定性，影响杠杆-手臂的动力学和有效性。由疾病相关的MHC或MLC候选突变修饰的肌球蛋白进行了体外和原位实验，以确定这些突变如何影响MHC中运行在天然转导工作模型中的功能结构域，或者MLC提供的杠杆-手臂稳定性。检测杠杆-手臂旋转运动的单分子实验特别相关，因为它适用于肌球蛋白在肌纤维天然拥挤的环境中。肌球蛋白调节轻链(RLC)可能具有特殊的意义，因为在正常心脏组织中，它在Ser15位部分被磷酸化。磷酸化明显影响肌球蛋白的钙调节，而在肌肉组织和肌球蛋白占空比的体外单个肌球蛋白马达。在后一种情况下，磷酸化的RLC构象调节必然影响与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调控肌球蛋白的结构基础，以及FHC连锁突变对RLC结构的影响。 公共卫生相关性：家族性肥厚型心肌病(FHC)是一种以心脏增大为特征的疾病。它影响500人中的1人，是年轻人心源性猝死的原因之一。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性化药物的目标。这项拟议的研究应用了前景看好的新的计算和实验方法来评估与疾病有关的突变如何改变运动动力收缩。

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.