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

描述（由申请人提供）：ryanodine受体1型（RyR1）通过释放兴奋-收缩耦合所需的Ca2+在骨骼肌收缩中起关键作用。这种巨大的同四聚体Ca2+通道的错义突变可导致衰弱性骨骼肌疾病，包括恶性高热（MH）。然而，RyR1大分子复合物的整体结构以及这些突变对该结构的影响在很大程度上是未知的。本提案的短期目标是开发一种创新的方法，利用Forester共振能量转移（FRET）技术来绘制RyR1的整体三级和四级结构。这种生物物理方法通过精确测量蛋白质中定义位置之间的距离来提供独特的结构信息。FRET可以用来确定当蛋白质改变构象时这些距离是如何变化的。这项工作的长期目标是使用这种方法绘制野生型和MH突变的RyR1，从而获得对该蛋白结构的新见解。假设：基于fret的实验系统由一个n端融合的GFP供体和一个针对RyR1初级序列中His标签的荧光受体组成，可用于测量RyR结构内的分子内距离。具体目的：我们将建立一种基于FRET的检测方法，通过将FRET供体和受体分子放置在该蛋白表面来测量RyR1定义的一级序列元件之间的距离。该FRET对将由一个n端融合的绿色荧光蛋白（GFP；荧光供体）和由PI合成的荧光受体（NTA-1和NTA-Rho）组成，这些荧光受体结合到战略性地放置在RyR1初级序列明确区域的多组氨酸标签上。从n端GFP到这些His标签的点对点距离将通过FRET计算，可以测量供体荧光的减少（对于NTA-1）或供体荧光猝灭以及受体荧光的增强（对于NTA-Rho）。我们将对GFP- RyR1融合蛋白进行基于细胞和体外的FRET测量，以估计邻近RyR1通道产生的FRET信号的贡献。展望：通过这一系列综合实验，我们将开发一套新的分子工具来定位特异性标记RyR1，然后使用FRET检查该蛋白的结构。这些实验将为未来的实验奠定基础，这些实验有可能提供RyR1结构的前所未有的一瞥，以及这种结构是如何被致病突变改变的。公共卫生相关性。该提案将建立一种基于fret的技术来可视化RyR1的结构。这项技术将为进一步测量RyR1的结构打开大门，以确定致病突变如何改变通道的结构。

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.