Conformational measurements of RyR1 using a site-specific labeling method

描述(申请人提供)：兰尼定受体1型(RyR1)通过释放兴奋-收缩(EC)偶联所需的钙离子，在骨骼肌收缩中发挥重要作用。RyR1中的一个关键结构域称为“钳区”，它在肌肉收缩过程中调节RyR1的激活。钳夹区域的错义突变可导致骨骼肌衰弱疾病，包括恶性高热(MH)和中枢性核心病(CCD)。然而，这个关键调控结构域的整体结构以及这些突变对RyR1激活过程中发生的构象变化的影响在很大程度上是未知的。这项建议的目的是利用一种创新的位点特异性标记方法与Forster共振能量转移(FRET)技术相结合来检测在完整细胞中表达的RyR1钳制区发生的构象变化。这项拟议工作的长期目标是比较野生型和MH/CD突变的RyR1中发生的构象变化，以确定由这些突变引起的RyR1的结构重排如何导致这些骨骼肌疾病。假设：RyR1的激活涉及钳夹区域内区域-结构域相互作用的变化，这些变化被导致骨骼肌疾病的点突变破坏。具体目的：在目标1中，将使用基于FRET的分析来测量在RyR1的前600个氨基酸中发现的3个假定亚域之间的构象变化。由融合到RyR1中的供体绿色荧光蛋白(GFP)和由P.I.(Cy3NTA)合成的针对插入RyR1一级序列中的10个残基组氨酸(His10)标签的荧光受体组成的FRET对将用于完整细胞和体外的FRET测量。在生理性(即EC偶联)和药物调节剂改变通道活性后，FRET的变化将表明供体和受体之间发生的构象变化。还将确定由于引入MH和CD突变而导致的这些构象运动的变化。在目标2中，这种FRET分析将被用于检验一个特定的结构“结构域切换”假说，该假说认为MH是由两个RyR1结构域(MH区1和2)之间的缺陷相互作用引起的。FRET从MH区1的GFP到插入MH区2的His10标签的变化的测量将被用来探索这两个结构域之间的这种可能的相互作用。观点：通过这一系列综合实验，一套新的分子工具将被用来研究与通道门控相关的蛋白质结构的动态变化，这些工具是为定位标记RyR1开发的。这些实验应该会让人们前所未有地一瞥RyR1的结构，以及这种结构是如何被致病突变所干扰的。 与公共健康相关：一种新的特定部位标记技术将用于可视化RyR1蛋白被激活时发生的结构变化。然后，通过使用这种方法来确定骨骼肌致病突变如何影响RyR1的这些结构变化，可以更全面地了解这些疾病的分子基础。

DESCRIPTION (provided by applicant): The ryanodine receptor type 1 (RyR1) plays a vital role in skeletal muscle contraction by releasing Ca2+ required for excitation-contraction (EC) coupling. A key structural domain in RyR1 called the "clamp region" mediates activation of RyR1 during muscle contraction. Missense mutations in the clamp region can cause debilitating skeletal muscle disorders including malignant hyperthermia (MH) and central core disease (CCD). However, the overall structure of this critical regulatory domain and the effects of these mutations on conformational changes that occur during RyR1 activation are largely unknown. The objective of this proposal is to utilize an innovative site-specific labeling method combined with Forster resonance energy transfer (FRET) techniques to examine conformational changes that occur in the clamp region of RyR1 expressed in intact cells. The long-term goal of the proposed work is to compare conformational changes occurring in wildtype and MH/CCD mutated RyR1 to determine how structural rearrangements of RyR1 caused by these mutations can lead to these skeletal muscle disorders. Hypothesis: RyR1 activation involves changes in domain-domain interactions within the clamp region that are disrupted by point mutations that lead to skeletal muscle disorders. Specific Aims: In Aim 1, a FRET-based assay will be used to measure conformational changes between 3 putative sub-domains found in the first 600 amino acids of RyR1. A FRET pair consisting of a donor, green fluorescent protein (GFP) fused into RyR1 and a fluorescent acceptor synthesized by the P.I. (Cy3NTA) targeted to 10 residue histidine (His10) tags inserted into the RyR1 primary sequence will be used for FRET measurements in intact cells and in vitro. Changes in FRET after alteration of channel activity by physiological (i.e. EC coupling) and pharmacological modulators will indicate conformational changes occurring between the donor and acceptor sites. Changes in these conformational movements resulting from introduction of MH and CCD mutations will also be determined. In Aim 2, this FRET assay will be used to examine a specific structural "domain switch" hypothesis that suggests that MH is caused by a defective interaction between two RyR1 domains (MH zone 1 and 2). Measurements of changes in FRET from GFP in MH Zone 1 to His10 tags inserted into MH zone 2 that occur during EC coupling or as the result of bioactive "domain peptides" or MH/CCD mutations will be used to probe this putative interaction between these two domains. Perspective: Through this integrated series of experiments, a new set of molecular tools developed to site-specifically label RyR1 will be used to examine dynamic changes in protein structure involved in channel gating. These experiments should provide unprecedented glimpses of the structure of RyR1 and how this structure is perturbed by disease-causing mutations. PUBLIC HEALTH RELEVANCE: A novel site-specific labeling technique will be used to visualize changes in the structure of RyR1 that occur when the protein is activated. Then, by using this method to determine how skeletal muscle disease-causing mutations affect these structural changes of RyR1, the molecular basis of these disorders can be more completely understood.