cMyBP-C: Molecular Mechanisms of Actomyosin Modulation

cMyBP-C对肌动球蛋白力发电系统发挥作用的分子机制在很大程度上仍不清楚。由于其相对于肌球蛋白的比例较低，并且位于粗肌丝的不同区域，我们将在本项目中确定cMyBP-C如何通过与有限数量的横桥相互作用或是否协同影响粗肌丝内的所有横桥来调节肌动球蛋白的发电。该项目作为动物（项目#3，Robbins）、整个心脏（Core B，Kass）和纤维（Core B，Palmer，Maughan）研究之间的生理桥梁。在目标#1中，我们将使用最先进的单分子生物物理技术（例如激光陷阱测定）来探测cMyBP-C对肌动球蛋白功能的影响，该肌动球蛋白功能沿着从项目#3（Robbins）中设计并在核心C（Robbins）中产生的转基因小鼠中分离的单个天然粗丝的长度。在目标#2中，我们将使用在核心C（Robbins）中产生的cMyBP-C的表达的N末端片段来探测这些片段对肌动蛋白和/或肌球蛋白的结合亲和力。因此，这些数据以及在项目#1（克雷格）、项目#3（罗宾斯）和核心B（帕尔默）中获得的数据将有助于定义cMyBP-C的特异性结合配偶体（即肌球蛋白和/或肌动蛋白），从而其特异性结合配偶体（即肌球蛋白和/或肌动蛋白）。 生理作用部位。结合运动性和激光陷阱测定，我们将确定是否CMyBP-C的N-末端限制肌球蛋白的附着率肌动蛋白或如果它直接影响肌球蛋白的固有分子力学和动力学。最后，在目标#3中，我们将描述磷酸化如何调节cMyBP-C作用。使用表达cMyBP C突变体的转基因小鼠模型（Core C，Robbins），所述突变体具有丙氨酸或天冬氨酸取代三种可磷酸化丝氨酸中的一种或多种，我们将使用天然粗蛋白酶链反应来确定磷酸化的功能重要性和每个位点的分级重要性。 含有突变cMyBP-C以及具有相同突变的N-末端片段的细丝。一旦确定了cMyBP-C的分子机制，在与cMyBP-C基因突变相关的心力衰竭病例中，可能有可能进行新的治疗或临床干预。

The molecular mechanism by which cMyBP-C exerts its effect on actomyosin force power generating system remains largely undefined. With its low ratio relative to myosin and it being located in distinct regions of the thick filament, we will determine in this Project how cMyBP-C's modulates actomyosin's power generation by either interacting with a limited population of crossbridges or whether it cooperatively affects all crossbridges within the thick filament. This project serves as a physiological bridge between the animal (Project #3, Robbins), whole heart (Core B, Kass) and fiber (Core B, Palmer, Maughan) studies. In Aim #1, we will use state-of-the-art single molecule biophysical techniques (e.g. laser trap assay) to probe the effect that cMyBP-C exerts on actomyosin function along the length a single native thick filament isolated from transgenic mice designed in Project #3 (Robbins) and produced in Core C (Robbins). In Aim #2, we will use expressed N-terminal fragments of cMyBP-C produced in Core C (Robbins) to probe the binding affinity of these fragments for actin and/or myosin. Thus, these data and that obtained in Project #1 (Craig), Project #3 (Robbins) and Core B (Palmer) will help define cMyBP-C's specific binding partners (i.e. myosin and/or actin) and thus its physiological site of action. In combination with motility and laser trap assays, we will determine if the N-terminus of CMyBP-C limits myosin's attachment rate to actin or if it directly affects myosin's inherent molecular mechanics and kinetics. Finally, in Aim #3 we will characterize how phosphorylation regulates cMyBP-C action. Using transgenic mouse models (Core C, Robbins) expressing cMyBP C mutants having alanine or aspartic acid substitutions for one or more of the three phosphorylatable serines, we will determine the functional importance of phosphorylation and the hierarchical importance of each site using native thick filaments containing mutant cMyBP-C as well as N-terminal fragments having the same mutations. Once the molecular mechanism of cMyBP-C is defined, the potential for novel therapeutics or clinical intervention may be possible in cases of heart failure associated with genetic mutations in cMyBP-C.