cMyBP-C: Molecular Mechanisms of Actomyosin Modulation

cMyBP-C作用于肌动球蛋白力发电系统的分子机制仍未明确。由于其相对于肌凝蛋白的比例较低，并且位于粗丝的不同区域，我们将在本项目中确定cMyBP-C如何通过与有限的交叉桥相互作用或是否协同影响粗丝内的所有交叉桥来调节肌动球蛋白的发电。该项目作为动物（项目#3，罗宾斯），全心脏（核心B，卡斯）和纤维（核心B，帕尔默，莫恩）研究之间的生理桥梁。在Aim #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，该功能沿着从项目#3（罗宾斯）设计并在核心C（罗宾斯）生产的转基因小鼠中分离出的单个天然粗丝的长度。在Aim #2中，我们将使用在Core C中产生的cMyBP-C的n端表达片段（Robbins）来探测这些片段与肌动蛋白和/或肌凝蛋白的结合亲和力。因此，这些数据以及在项目#1 （Craig）、项目#3 （Robbins）和核心B （Palmer）中获得的数据将有助于定义cMyBP-C的特定结合伙伴（即肌凝蛋白和/或肌动蛋白），从而确定其

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.