Molecular mechanisms of RyR activation by 4-CmC

4-CmC 激活 RyR 的分子机制

• 批准号: 7106356
• 负责人: JAMES D FESSENDEN
• 金额: $ 12.97万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7106356
• 关键词: binding sites; biophysics; calcium channel; conformation; fluorescence resonance energy transfer; fluorescent dye /probe; genetic markers; high performance liquid chromatography; malignant hyperthermia; mass spectrometry; matrix assisted laser desorption ionization; muscle disorders; muscle function; muscle proteins; point mutation; posttranslational modifications; protein isoforms; protein sequence; protein structure function; striated muscles

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.

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.该应用程序的成功资金将有助于Fessenden博士从指导的科学家到能够通过自己的RO1应用程序获得资金的独立研究人员的过渡。 培训计划：培训计划将包括两个部分；对基于蛋白质化学的技术进行了指导的培训，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的指导。这项培训将由波士顿生物医学研究所（BBRI）的赞助商Noriaki Ikemoto博士进行监督。 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来研究RYR的构象机制。此外，由Paul Leavis博士，Renne Chen Lu博士和BBRI的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开发蛋白质化学/分子生物学的新技能。 环境：申请人将主要在BBRI上接受培训，这是一种最先进的研究机构：使用生化和生物物理技术研究肌肉蛋白的研究机构。此外，申请人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：该提案将检查骨骼肌ryanodine受体同工型（RYR1）上的关键调节器位点。调节剂4-氯-M-Cresol（4-CMC）是一种与临床相关的RYR激活剂，用于诊断骨骼肌疾病，恶性高温（MH）。使用分子生物学和生化技术，4-CMC的结合位点将位于RYR1主要序列中。此外，将使用构型敏感的荧光探针确定含有点突变的RYR1通道增强的4-CMC灵敏度的机制。最后，将使用荧光共振能传递（FRET）测量从4-CMC结合位点到RYR1上高亲和力ryanodine结合位点的分子距离。这项研究将对RYR的4-CMC激活的生物物理特征进行全面检查，同时也是使用基于FRET的技术进行更广泛探索RYR的出发点。