In Situ Sensing of Single Myosin Function in Hypertrophy Disease

描述（由申请人提供）：基因筛查检测到大量的肉瘤蛋白突变，阐明疾病的基本原因，并在蛋白质水平上发现功能缺陷时确定个体化药物的靶点。该项目专注于运动肌球蛋白分子及其调控，使用各种方法对其天然和突变形式的蛋白质进行表达、动力学表征和结构可视化。目的是破译个体突变在修改天然肌凝蛋白功能中所起的作用。肌球蛋白通过调节活性位点的ATP水解、肌动蛋白结合位点的肌动蛋白亲和调节和杠杆臂的力量行程，通过时间顺序的变构转导途径，将ATP自由能转导为机械功。能量转导是肌球蛋白的决定性系统特征，天然转导的工作模型将特定功能分配到马达内的结构域，从活性部位的ATP水解开始，到在肌肉组织拥挤的环境中旋转马达中的杠杆臂结构域70度的动力行程结束。心肌肌球蛋白重链（MHC）及其两条轻链（mlc）携带家族性肥厚性心肌病（FHC）相关突变。假设MHC突变体破坏特定的转导途径。进化上保守的变构连接预测识别MHC中形成转导途径的残基。转导途径残基也是fhc连锁突变位点，确定影响转导的MHC候选突变体。假设几种MLC突变体影响杆臂结构稳定性，影响杆臂动力学和有效性。由疾病相关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MHC在天然转导的工作模型中运作的功能域，或MLC提供的杠杆臂稳定性。检测杠杆臂旋转运动的单分子实验尤其有意义，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（Myosin regulatory light chain， RLC）可能具有特殊意义，因为它在正常心脏组织中的Ser15位点部分磷酸化。磷酸化明显影响肌凝蛋白钙的调节，而在肌肉组织和肌凝蛋白在体外单个肌凝蛋白马达的占空比。在后一种情况下，RLC构象的磷酸化调节必须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调控肌球蛋白的结构基础以及fhc连锁突变对RLC结构的影响。

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.