Molecular mechanisms of RyR activation by 4-CmC

描述(申请人提供)：申请者：James D.Fessenden博士是波士顿生物医学研究所(BBRI)的讲师。在他的博士后工作中，Fessenden博士使用细胞/分子生物学技术来确定骨骼肌钙释放通道的结构成分-ryanodine受体(RyR)-如何对其功能做出贡献。这项申请的成功资助将帮助费森登博士从一名受指导的科学家转变为一名能够通过自己的RO1申请获得资金的独立研究员。 培训计划：培训计划将由两部分组成：以蛋白质化学为基础的技术指导培训，以研究RyR结构-功能；以及正式课程，以扩大指导培训，并就负责任的研究行为提供指导。本次培训将由赞助商波士顿生物医学研究所(BRI)的Noriaki Ikemoto博士监督。Ikemoto博士是RyR结构生物学领域的著名研究员，他开创了使用荧光探针来研究RyR构象机制的先河。此外，由一带一路的Paul Leavis博士、Renne陈露博士和Sherwin S.Lehrer博士以及哈佛医学院的Paul Allen博士组成的杰出咨询委员会将帮助候选人发展蛋白质化学/分子生物学方面的新技能。 环境：申请者将主要在BBRI接受培训，BBRI是一家致力于使用生化和生物物理技术研究肌肉蛋白质的最新研究机构。此外，申请者将可以使用位于哈佛医学院的艾伦实验室提供的设施和资源。 研究计划：这项提案将研究骨骼肌Ryanodine受体亚型(RyR1)上的一个关键调控位点。调节剂4-氯-间甲酚(4-CMC)是一种临床相关的RyR激活剂，用于诊断患有骨骼肌疾病的恶性高热(MH)。利用分子生物学和生化技术，4-CMC的结合位点将定位在RyR1的一级序列中。此外，包含导致MH的点突变的RyR1通道的4-CMC敏感性增强的机制将使用构象敏感的荧光探针来确定。最后，将使用荧光共振能量转移(FRET)测量从4-CMC结合部位到RyR1上高亲和力ryanodine结合部位的分子距离。这项研究将全面检查4-CMC激活RyR的生物物理特征，同时也是使用基于FRET的技术更广泛地探索RyR的起点。

DESCRIPTION (provided by applicant): Applicant: Dr. James D. Fessenden is an Instructor at the Boston Biomedical Research Institute (BBRI) for his postdoctoral work, Dr. Fessenden has used cell/molecular biology techniques to determine how structural components of the skeletal muscle Ca -release channel, the ryanodine receptor (RyR), contribute to its function. The successful funding of this application will aid Dr. Fessenden in the transition from a mentored scientist to an independent investigator capable of obtaining funding through his own RO1 application. Training Plan: The training plan will consist of two parts; mentored training on protein chemistry-based techniques to study RyR structure-function and formal coursework to amplify the mentored training and provide instruction on the responsible conduct of research. This training will be supervised by the Sponsor, Dr. Noriaki Ikemoto at the Boston Biomedical Research Institute (BBRI). Dr. Ikemoto, a renowned researcher in the field of RyR structural biology, has pioneered the use of fluorescent probes to study conformational mechanisms of the RyR. In addition, a distinguished Advisory Committee consisting of Dr. Paul Leavis, Dr. Renne Chen Lu and Dr. Sherwin S. Lehrer at the BBRI as well as Dr. Paul Allen at the Harvard Medical School will assist the Candidate in developing new skills in protein chemistry/molecular biology. Environment: The Applicant will be trained primarily at the BBRI, a state-of-the-ar: research facility devoted to the study of muscle proteins using biochemical and biophysical techniques. In addition, the Applicant will have access to the facilities and resources offered by the Allen lab located at the Harvard Medical School. Research Plan: This proposal will examine a key modulator site on the skeletal muscle ryanodine receptor isoform (RyR1). The modulator, 4-chloro-m-cresol (4-CmC), is a clinically relevant RyR activator used to diagnose patjents with the skeletal muscle disorder, malignant hyperthermia (MH). Using molecular biology and biochemical techniques, the binding site for 4-CmC will be localized within the RyR1 primary sequence. In addition, the mechanism of enhanced 4-CmC sensitivity of RyR1 channels containing point mutations that result in MH will be determined using conformationally sensitive fluorescent probes. Finally, molecular distances from the 4-CmC binding site to the high affinity ryanodine binding site on RyR1 will be measured using fluorescence resonance energy transfer (FRET). This study will provide a comprehensive examination of the biophysical characteristics of 4-CmC activation of the RyR while also serving as a point of departure for broader explorations of the RyR using FRET-based techniques.