A FRET-based assay to measure dynamic changes in RyR1 structure

基于 FRET 的测定法测量 RyR1 结构的动态变化

• 批准号: 7512031
• 负责人: JAMES D FESSENDEN
• 金额: $ 26.8万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7512031
• 关键词: Abbreviations; Acids; Address; Affinity; Binding; Biological Assay; Cells; Central Core Myopathy; Chimeric Proteins; Complex; Coupling; Critiques; Data; Data Analyses; Edetic Acid; Elements; Energy Transfer; FUS-1 Protein; Fluorescence; Funding; Funding Mechanisms; Future; Green Fluorescent Proteins; Guidelines; Histidine; In Vitro; Label; Macromolecular Complexes; Malignant hyperpyrexia due to anesthesia; Maps; Measurement; Measures; Membrane Proteins; Methodology; Methods; Missense Mutation; Molecular; Molecular Conformation; Muscle Contraction; Mutate; Mutation; Myopathy; N-terminal; Placement; Play; Positioning Attribute; Preparation; Protein Isoforms; Proteins; Public Health; Purpose; Rate; Reagent; Reporting; Research Project Grants; Role; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Series; Signal Transduction; Site; Skeletal system; Staging; Structure; Study Section; System; Techniques; Testing; Thin Layer Chromatography; United States National Institutes of Health; Vesicle; Work; base; disease-causing mutation; his6 tag; in vivo; innovation; insight; novel; protein structure; research study; rho; stoichiometry; tetramethylrhodamine; tool

DESCRIPTION (provided by applicant): The ryanodine receptor type 1 (RyR1) plays a critical role in skeletal muscle contraction by releasing the Ca2+ required for excitation-contraction coupling. Missense mutations in this enormous homotetrameric Ca2+ channel can cause debilitating skeletal muscle disorders including malignant hyperthermia (MH). However, the overall structure of the RyR1 macromolecular complex and the effects of these mutations on this structure are largely unknown. The short-term objective of this proposal is to develop an innovative method to map the overall tertiary and quaternary structure of RyR1 using Forester resonance energy transfer (FRET) techniques. This biophysical method provides unique structural information by accurately measuring distances between defined positions in the protein. FRET can then be used to determine how these distances change when the protein changes conformation. The long-term objective of the proposed work is to map both wild type and MH mutated RyR1 using this method and thereby gain novel insights into the structure of this protein. Hypothesis: A FRET-based experimental system comprised of an N-terminally fused GFP donor and a fluorescence acceptor targeted to His tags in the primary sequence of RyR1 can be used to measure intramolecular distances within the RyR structure. Specific Aim: We will establish a FRET-based assay to measure distances between defined primary sequence elements of RyR1 by placing both a FRET donor and an acceptor molecule onto the surface of this protein. This FRET pair will consist of an N-terminally fused green fluorescent protein (GFP; fluorescence donor) and fluorescence acceptors (NTA-1 and NTA-Rho) synthesized by the PI that bind to poly-histidine tags strategically placed into well-defined regions of the RyR1 primary sequence. Point to point distances from the N-terminal GFP to these His tags will be computed from FRET measured either as a decrease in donor fluorescence (for NTA-1) or both donor fluorescence quenching as well as enhancement of acceptor fluorescence (for NTA-Rho). We will conduct both cell-based as well as in vitro FRET measurements of GFP- RyR1 fusion proteins in order to estimate the contribution of FRET signals arising from neighboring RyR1 channels. Perspective: Through this integrated series of experiments, we will develop a new set of molecular tools to site-specifically label RyR1 and then examine the structure of this protein using FRET. These experiments will set the stage for future experiments that have the potential to provide unprecedented glimpses of the structure of RyR1 and how this structure is changed by disease-causing mutations. PUBLIC HEALTH RELEVANCE. The proposal will establish a FRET-based technique to visualize the structure of RyR1. This technique will open the door for further structural measurements of RyR1 to determine how disease-causing mutations alter the structure of the channel.

描述（由申请人提供）：ryanodine受体1型（RYR1）通过释放激发反应耦合所需的Ca2+，在骨骼肌收缩中起关键作用。这种巨大的同型Ca2+通道中的错义突变会导致衰弱的骨骼肌疾病，包括恶性高温（MH）。但是，RYR1大分子复合物的总体结构以及这些突变对该结构的影响在很大程度上尚不清楚。该提案的短期目标是开发一种创新的方法，使用Forester Resonance Enermance Transniques（FRET）技术来绘制RYR1的整体第三和第四纪结构。这种生物物理方法通过准确测量蛋白质定义位置之间的距离来提供独特的结构信息。然后可以使用FRET来确定当蛋白质改变构象时，这些距离如何变化。拟议工作的长期目标是使用此方法同时绘制野生型和MH突变的RYR1，从而获得对该蛋白质结构的新见解。假设：一个基于FRET的实验系统，该系统由N端融合的GFP供体组成，以及针对其标签的RyR1序列中的标签的荧光受体，可用于测量RYRR结构内的分子内距离。具体目的：我们将通过将FRET供体和受体分子同时将基于FRET的测定方法建立，以测量RYR1定义的主要序列元素之间的距离。该FRET对将由由PI合成的N末端融合的绿色荧光蛋白（GFP；荧光供体）和荧光受体（NTA-1和NTA-RHO）组成，该蛋白（NTA-1和NTA-RHO）由PI合成，该PI结合了策略性地置于Ryr1主要序列的策略性地定义的区域。从N末端GFP到这些标签的点到点距离将是根据供体荧光降低（对于NTA-1）的降低或供体荧光猝灭以及增强受体荧光的增强（对于NTA-RHO）的供体荧光（用于NTA-RHO）。我们将对GFP-RYR1融合蛋白进行基于细胞的基于细胞的FRET测量，以估计邻近RYR1通道引起的FRET信号的贡献。透视图：通过一系列集成的实验，我们将开发一组新的分子工具来特异性标记RYR1，然后使用FRET检查该蛋白质的结构。这些实验将为将来的实验奠定阶段，这些实验有可能对RYR1结构进行前所未有的瞥见以及如何通过引起疾病的突变改变这种结构。 公共卫生相关性。 该提案将建立一种基于FRET的技术，以可视化RYR1的结构。该技术将为RYR1进行进一步的结构测量打开大门，以确定引起疾病的突变如何改变通道的结构。