Conformational measurements of RyR1 using a site-specific labeling method

描述（由申请人提供）：ryanodine受体1型（RyR1）通过释放兴奋-收缩（EC）耦合所需的Ca2+在骨骼肌收缩中起重要作用。RyR1的一个关键结构域被称为“钳区”，在肌肉收缩期间介导RyR1的激活。钳区错义突变可导致衰弱性骨骼肌疾病，包括恶性高热（MH）和中央核心疾病（CCD）。然而，这个关键调控区域的整体结构以及这些突变对RyR1激活期间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）技术来检查完整细胞中表达的RyR1的钳位区发生的构象变化。这项工作的长期目标是比较野生型和MH/CCD突变的RyR1中发生的构象变化，以确定这些突变引起的RyR1结构重排如何导致这些骨骼肌疾病。假设：RyR1激活涉及钳位区域内结构域-结构域相互作用的变化，该区域被导致骨骼肌疾病的点突变破坏。具体目的：在目标1中，基于fret的检测将用于测量RyR1前600个氨基酸中3个假定亚结构域之间的构象变化。FRET对包括一个供体，绿色荧光蛋白（GFP）融合到RyR1和一个荧光受体合成的P.I. （Cy3NTA）靶向10个残基组氨酸（His10）标签插入到RyR1初级序列将用于FRET测量在完整细胞和体外。生理（即EC偶联）和药理学调节剂改变通道活性后FRET的变化将表明供体和受体位点之间发生构象变化。这些构象运动的变化也将由引入MH和CCD突变而确定。在目标2中，FRET实验将用于检查一个特定的结构“结构域切换”假设，该假设表明MH是由两个RyR1结构域（MH区1和2）之间有缺陷的相互作用引起的。测量从MH 1区GFP到插入MH 2区His10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假定的相互作用。观点：通过这一系列的综合实验，一套新的分子工具将被开发出来，用于特异性标记RyR1，以检测参与通道门控的蛋白质结构的动态变化。这些实验将为RyR1的结构以及这种结构如何受到致病突变的干扰提供前所未有的一瞥。

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.