Structural Studies of RyR Channel

RyR通道的结构研究

• 批准号: 8507907
• 负责人: Irina I Serysheva
• 金额: $ 20.58万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8507907
• 关键词: Address; Amino Acid Sequence; Architecture; Brain; Buffers; Cell physiology; Complex; Computer software; Cryoelectron Microscopy; Cytoplasm; Data Collection; Defect; Detergents; Disease; Drug Design; Environment; Future; Generations; Goals; Heart; Human; Ice; Image; Integral Membrane Protein; Ion Channel; Ligands; Lipids; Liposomes; Location; Malignant hyperpyrexia due to anesthesia; Mediating; Membrane; Membrane Lipids; Membrane Proteins; Methodology; Methods; Molecular; Molecular Conformation; Muscle Contraction; Muscle Fibers; Myopathy; Nature; Peptide Sequence Determination; Pharmaceutical Preparations; Play; Polymers; Preparation; Proteins; Publishing; Research; Resolution; Role; RyR1; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcoplasmic Reticulum; Signal Transduction; Skeletal Muscle; Solutions; Specimen; Structure; Therapeutic; Thick; Transmembrane Domain; Variant; Water; X-Ray Crystallography; aqueous; base; computerized tools; density; improved; in vitro Assay; insight; particle; protein complex; public health relevance; reconstitution; reconstruction; surfactant; three dimensional structure; unilamellar vesicle

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.

描述(申请人提供)：1型Ryanodine受体(RyR1)是骨骼肌中一种细胞内配体门控的钙释放通道，它负责导致肌肉收缩的胞浆游离钙浓度增加。这一关键蛋白的缺陷或其活性的调节导致肌浆网钙离子的异常动员，导致多种肌肉疾病，如恶性高热、中央核心和多微核心疾病。目前缺乏RyR1的高分辨率结构限制了我们理解这一通道在自然和疾病状态下如何发挥作用的能力。由于RyRs的大小(~2.3 mda)、位于膜环境中和动态性质，对RyRs的结构分析具有极大的挑战性。到目前为止，单颗粒冷冻-EM是分析如此大的完整膜蛋白结构的最可行的方法。然而，由于低温冷冻液中存在洗涤剂，对RyRs的冷冻-EM研究面临着另一个障碍。这在优化冰层厚度和EM成像以产生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。通常，降低的图像对比度是产生高分辨率膜蛋白结构的主要障碍。利用低温EM实现RyR1的高分辨率结构显然需要在低温样品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单颗粒冷冻-EM分析的RyR1玻璃化方法，该方法使用一种新的表面活性剂，两亲性聚合物，来代替洗涤剂 使通道蛋白在无洗涤剂的水溶液中溶解并保持其功能形式(AIM1)。通过使用这种方法，我们有望在低温电子显微镜研究中避开洗涤剂带来的困难，并在亚纳米分辨率下实现封闭状态下RyR1的三维重建。然后，我们建议将RyR1通道重构为小的单层囊泡，并使用单粒子重构的变体来解决脂质膜中的通道结构(目标2)。将在EMAN/EMAN2软件的框架内为该项目开发新的计算工具，以便在当前~1 nm以外的分辨率下实现完整的RyR1的结构，并在膜环境中建立其三维结构。确定的结构将揭示具有机械信息的蛋白质特征，这将使人们能够对RyR1通道功能有重要的了解。一旦建立了最佳的方法，我们预计将把RyR1的结构分析扩展到近原子分辨率和不同的生理相关功能状态。作为这项研究的一部分，所开发的方法将对其他离子通道和近天然状态下的大型膜蛋白复合体的研究具有广泛的适用性。