Structural Studies of RyR Channel

描述（由申请人提供）：1型兰尼碱受体（RyR 1）是骨骼肌中的一种细胞内配体门控Ca 2+释放通道，负责增加游离细胞质Ca 2+浓度，导致肌肉收缩。这种关键蛋白质或其活性调节的缺陷导致肌浆网的异常Ca 2+动员，导致几种肌肉疾病，如恶性高热、中央核心和多微核心疾病。目前缺乏RyR 1的高分辨率结构限制了我们了解该通道在天然和疾病状态下如何发挥作用的能力。RyR的结构分析是非常具有挑战性的，因为它们的大尺寸（~2.3 MDa），在膜环境中的位置和动态性质。迄今为止，单颗粒冷冻EM是最可行的方法结构分析，这样的大的完整的膜蛋白。然而，冷冻电镜研究RyRs面临一个额外的障碍，由于存在的洗涤剂在冷冻标本。这在优化冰厚度和EM成像以生成具有良好对比度的图像以进行可靠的3D重建方面提出了许多问题。降低的图像对比度通常是产生膜蛋白的高分辨率结构的主要障碍。通过冷冻EM实现RyR 1的高分辨率结构显然需要在冷冻标本制备和成像条件的方法学上取得突破。在这个项目中，我们的目标是开发一种玻璃化RyR 1的方法，用于单颗粒冷冻EM分析，该方法利用一类新的表面活性剂，两亲聚合物，代替去污剂， 保持通道蛋白在无洗涤剂的水溶液（AIM 1）中可溶并处于其功能形式。通过使用这种方法，我们预期规避洗涤剂施加的困难，在冷冻EM研究，并实现在亚纳米分辨率的封闭状态下的RyR 1的三维重建。然后，我们提出将RyR 1通道重建成小的单层囊泡，并使用单颗粒重建的变体来解决脂质膜中的通道结构（目的2）。将在EMAN/EMAN 2软件的框架内为该项目开发新的计算工具，以便在超过当前约1 nm的分辨率下实现完整RyR 1的结构，并在膜环境中建立其3D结构。确定的结构将揭示机械信息蛋白质的功能，将允许重要的见解RyR 1通道功能。一旦建立了最佳的方法，我们预计将RyR 1的结构分析扩展到近原子分辨率和不同的生理相关的功能状态。作为本研究的一部分，所开发的方法将具有广泛的适用性，以研究其他离子通道和大的膜蛋白复合物在近天然状态。

DESCRIPTION (provided by applicant): Type 1 ryanodine receptor (RyR1) is an intracellular ligand-gated Ca2+ release channel in skeletal muscle where it is responsible for the increase of free cytoplasmic Ca2+ concentration leading to muscle contraction. Defects in this key protein or in modulation of its activity cause aberrant Ca2+ mobilization from the sarcoplasmic reticulum resulting in several muscle disorders such as Malignant Hyperthermia, Central Core and Multi-minicore Diseases. Lack of high-resolution structure of RyR1 currently limits our ability to understand how this channel functions both in native and disease states. The structural analysis of RyRs is exceptionally challenging due to their large size (~2.3 MDa), location in the membrane environment and dynamic nature. To date, single particle cryo-EM is the most viable methodology for structural analysis of such large integral membrane proteins. However, cryo-EM studies of RyRs confront an additional hurdle due to the presence of detergent in cryospecimen. This raises a number of issues with optimization of ice thickness and EM imaging to produce images with a good contrast for reliable 3D reconstruction. The reduced image contrast is the main impediment to producing high-resolution structures of membrane proteins in general. Achieving a high- resolution structure of RyR1 by cryo-EM clearly requires a breakthrough in methodology for cryo-specimen preparation and imaging conditions. In this project, we aim to develop a method for vitrification of RyR1 for single particle cryo-EM analysis that utilizes the use of a new class of surfactants, amphipathic polymers, in place of detergent to keep the channel protein soluble and in its functional form in aqueous detergent-free solution (aim1). By using this approach we anticipate to circumvent detergent-imposed difficulties in cryo-EM studies and to achieve the 3D reconstruction of RyR1 in a closed state at subnanometer resolution. We then propose to reconstitute RyR1 channel into small unilamellar vesicles and to use a variant of single particle reconstruction to solve the channel structure in the lipid membrane (aim 2). New computational tools will be developed for this project within the framework of EMAN/EMAN2 software in order to achieve the structure of intact RyR1 at resolutions beyond the current ~1 nm and to establish its 3D architecture in membrane environment. The determined structures will reveal mechanistically informative protein features that will allow important insights into RyR1 channel function. Once optimal methodologies are established, we anticipate to extend the structural analysis of RyR1 to near-atomic resolution and to different physiologically relevant functional states. The methods developed, as part of this research, will have broad applicability to studies of other ion channels and large membrane protein complexes in near-native state.