In Situ Sensing of Single Myosin Function in Hypertrophy Disease

描述（由申请人提供）：基因筛选已检测到肌节蛋白中的大量突变，阐明了疾病的基本原因，并在可鉴定蛋白质水平上的功能缺陷时鉴定个体化药物的靶点。该项目的重点是分子马达肌球蛋白及其调节使用各种方法的表达，动力学表征和结构可视化的蛋白质在其天然和突变的形式。目标是破译单个突变在修饰天然肌球蛋白功能中所起的作用。肌球蛋白通过在活性位点协调ATP水解、在肌动蛋白结合位点协调肌动蛋白亲和力调节以及通过按时间顺序操作的变构转导途径进行双臂动力冲程，从而将ATP自由能转导成机械功。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给马达内的结构域，开始于活性位点中的ATP水解，结束于动力冲程，在肌肉组织的拥挤环境中将马达中的杠杆臂域旋转约70度。心肌肌球蛋白重链（MHC）及其两条轻链（MLCs）具有家族性肥厚型心肌病（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。也是FHC连锁突变位点的转导途径残基鉴定影响转导的MHC候选突变体。假设几种MLC突变体影响了横臂的结构稳定性，从而影响横臂的动力学和有效性。对由疾病相关的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的工作模型中操作的MHC中的功能结构域或MLC提供的单臂稳定性。检测前臂旋转运动的单分子实验是特别相关的，因为它适用于肌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的Ser15部分磷酸化。磷酸化明显影响肌球蛋白钙调节，而在肌肉组织和肌球蛋白占空比在体外单一肌球蛋白马达。在后一种情况下，RLC构象调制磷酸化必须影响肌球蛋白功能相关的强肌动蛋白结合。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.