Research supplement to promote diversity in Heath-related research

摘要 本研究补充的目的是通过直接检查 形成心肌肌球蛋白的抑制状态，即超松弛状态（SRX），并检查 cMyBP-C和疾病突变对这种关键结构状态的形成/稳定的影响。的重要性 研究表明，cMyBP-C在心肌收缩力中的作用， 磷酸化状态在增强收缩性中起作用，并且在心力衰竭期间磷酸化降低 可能导致收缩缺陷。cMyBP-C被认为影响心肌肌球蛋白SRX， 磷酸化依赖方式。在这个建议中，我们将设计一个FRET生物传感器的SRX， 允许直接检查cMyBP-C对肌球蛋白的这种至关重要的构象的影响。我们将 我们还研究了心肌肌球蛋白S2区的肥厚型心肌病（HCM）突变， cMyBP-C在目标1中，我们将通过将FRET信号与其他信号相关联来表征FRET生物传感器。 SRX的测量，如肌动蛋白激活的ATP酶和单转换ATP酶测定。我们会有所不同 离子强度，并检查药物Omecamtiv Mercabil，以证明FRET传感器可以用于 来测量SRX构象中心肌肌球蛋白的摩尔分数。在目标2中，我们将介绍HCM 突变进入S2区，并检查它们对SRX形成/稳定的影响。最后我们将 还检查cMyBP-C是否可以改变HCM突变体对SRX的形成/稳定的响应。总体看 该提案将通过提供肌球蛋白结构的直接测量来大大补充母体R 01， cMyBP-C在肌肉粗细丝调节中的作用研究将是至关重要的 收缩。 目标1。通过FRET检查人心肌肌球蛋白超松弛状态（SRX）的形成。 目标#2。检查心肌肌球蛋白S2区HCM突变的功能影响。

Abstract The goal of this research supplement is to complement the aims of the parent R01 by directly examining the formation of the inhibited state of cardiac myosin, super-relaxed state (SRX), and to examine the influence of cMyBP-C and disease mutations on the formation/stabilization of this key structural state. The importance of understanding the role of cMyBP-C in cardiac contractility is highlighted by work demonstrating that its phosphorylation state plays a role in enhancing contractility and during heart failure decreased phosphorylation likely contributes to contractile defects. cMyBP-C is proposed to influence the cardiac myosin SRX in a phosphorylation dependent manner. In this proposal we will design a FRET biosensor of the SRX which will allow direct examination of the influence of cMyBP-C on this crucially important conformation of myosin. We will also examine hypertrophic cardiomyopathy (HCM) mutations in the cardiac myosin S2 region known to interact with cMyBP-C. In Aim 1 we will characterize the FRET biosensor by correlating the FRET signal with other measurements of the SRX, such as actin-activated ATPase and single turnover ATPase assays. We will vary the ionic strength and examine the drug Omecamtiv Mercarbil to demonstrate that the FRET sensor can be used to measure the mole fraction of cardiac myosin in the SRX conformation. In Aim 2 we will introduce HCM mutations into the S2 region and examine their impact on the formation/stabilization of the SRX. Finally we will also examine if cMyBP-C can alter the HCM mutants response to formation/stabilization of the SRX. Overall, the proposal will greatly complement the parent R01 by providing direct measurements of myosin structure which will be crucial for interpreting the studies of cMyBP-C role in thick and thin filament regulation of muscle contraction. AIM #1. Examine the formation of the super-relaxed state (SRX) by FRET in human cardiac myosin. AIM #2. Examine the functional impact of HCM mutations in the S2 region of cardiac myosin.