Structural Studies of RyR Channel

描述（由申请人提供）：1型ryanodine受体（RyR1）是骨骼肌中细胞内配体门控的Ca2+释放通道，在那里它负责增加游离细胞质Ca2+浓度导致肌肉收缩。这种关键蛋白的缺陷或其活性的调节导致肌浆网Ca2+的异常动员，导致多种肌肉疾病，如恶性高热、中央核心和多小核心疾病。目前缺乏高分辨率的RyR1结构限制了我们了解该通道在原生和疾病状态下的功能。由于RyRs的大尺寸（~2.3 MDa），在膜环境中的位置和动态性质，结构分析非常具有挑战性。迄今为止，单颗粒低温电镜是最可行的方法，以结构分析这种大的整体膜蛋白。然而，由于冷冻样品中洗涤剂的存在，ryr的低温电镜研究面临着一个额外的障碍。这就提出了一些优化冰厚度和EM成像的问题，以产生具有良好对比度的图像，从而实现可靠的3D重建。一般来说，图像对比度降低是产生高分辨率膜蛋白结构的主要障碍。通过冷冻电镜获得RyR1的高分辨率结构显然需要在冷冻标本制备方法和成像条件方面取得突破。在这个项目中，我们的目标是开发一种用于RyR1单颗粒低温电镜分析的玻璃化方法，该方法利用了一类新的表面活性剂，两亲性聚合物来代替洗涤剂

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.