Conformational measurements of RyR1 using a site-specific labeling method

描述（由申请人提供）：1型兰尼碱受体（RyR 1）通过释放兴奋-收缩（EC）偶联所需的Ca 2+在骨骼肌收缩中发挥重要作用。RyR 1中的一个关键结构域称为“钳位区”，在肌肉收缩期间介导RyR 1的激活。错义突变的钳区域可以导致衰弱的骨骼肌疾病，包括恶性高热（MH）和中央核心疾病（CCD）。然而，这个关键的调控结构域的整体结构和这些突变对RyR 1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。本提案的目的是利用一种创新的位点特异性标记方法结合福斯特共振能量转移（FRET）技术来检查在完整细胞中表达的RyR 1的钳位区域中发生的构象变化。拟议工作的长期目标是比较野生型和MH/CCD突变RyR 1中发生的构象变化，以确定由这些突变引起的RyR 1结构重排如何导致这些骨骼肌疾病。假设：RyR 1激活涉及钳夹区域内结构域-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。具体目标：在目标1中，将使用基于FRET的测定来测量在RyR 1的前600个氨基酸中发现的3个推定亚结构域之间的构象变化。FRET对由供体、融合到RyR 1中的绿色荧光蛋白（GFP）和由P.I.靶向插入RyR 1一级序列的10个残基组氨酸（His 10）标签的Cy 3 NTA将用于完整细胞和体外FRET测量。通过生理学（即EC偶联）和药理学调节剂改变通道活性后FRET的变化将指示供体和受体位点之间发生的构象变化。还将确定由MH和CCD突变引入引起的这些构象运动的变化。在目标2中，该FRET测定将用于检查特定结构的“结构域转换”假设，该假设表明MH是由两个RyR 1结构域（MH区1和2）之间的缺陷相互作用引起的。测量从MH区1中的GFP到插入MH区2中的His 10标签的FRET变化，这些变化发生在EC偶联期间或作为生物活性“结构域肽”或MH/CCD突变的结果，将用于探测这两个结构域之间的这种推定的相互作用。视角：通过这一系列综合实验，开发了一套新的分子工具，用于位点特异性标记RyR 1，将用于研究参与通道门控的蛋白质结构的动态变化。这些实验应该提供前所未有的RyR 1结构的一瞥，以及这种结构是如何被致病突变扰乱的。

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.