Molecular mechanisms of RyR activation by 4-CmC

描述（由申请人提供）：申请人：James D.费森登博士是波士顿生物医学研究所（BBRI）博士后工作的讲师，费森登博士使用细胞/分子生物学技术来确定骨骼肌钙释放通道的结构成分兰尼碱受体（RyR）如何促进其功能。该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RO 1申请获得资助的独立研究者。 培训计划：该培训计划将包括两个部分;基于蛋白质化学技术的指导培训，以研究RyR结构-功能和正式课程，以扩大指导培训，并提供有关负责任的研究行为的指导。该培训将由申办者，波士顿生物医学研究所（BBRI）的Noriaki Ikemoto博士监督。Ikemoto博士是RyR结构生物学领域的著名研究人员，他率先使用荧光探针研究RyR的构象机制。此外，由Paul Leavis博士、Renne Chen Lu博士和Sherwin S. BBRI的Lehrer和哈佛医学院的Paul艾伦博士将协助候选人发展蛋白质化学/分子生物学方面的新技能。 工作环境：申请人将主要在BBRI接受培训，BBRI是一家致力于使用生物化学和生物物理技术研究肌肉蛋白的研究机构。此外，申请人将有权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：该提案将检查骨骼肌ryanodine受体亚型（RyR 1）的关键调节位点。调节剂4-氯-间甲酚（4-CmC）是临床上相关的RyR激活剂，用于诊断患有骨骼肌疾病（恶性高热（MH））的患者。使用分子生物学和生物化学技术，4-CmC的结合位点将位于RyR 1一级序列内。此外，含有导致MH的点突变的RyR 1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，使用荧光共振能量转移（FRET）测量从4-CmC结合位点到RyR 1上的高亲和力ryanodine结合位点的分子距离。这项研究将提供一个全面的检查4-CmC激活的RyR的生物物理特性，同时也作为一个出发点，更广泛的探索RyR使用FRET为基础的技术。

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.