Structural Studies of RyR Channel

RyR通道的结构研究

基本信息

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
项目类别：
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

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

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项目摘要

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（由申请人提供）：1型Ryanodine受体（RYR1）是骨骼肌中的细胞内配体门控Ca2+释放通道，在该通道中，它负责增加自由细胞质Ca2+浓度，从而导致肌肉收缩。该关键蛋白质的缺陷或对其活性的调节导致肌浆网的异常CA2+动员，导致多种肌肉疾病，例如恶性高温，中心核心和多米尼科氏病。当前，缺乏RYR1的高分辨率结构限制了我们了解该渠道在天然和疾病状态中如何发挥作用的能力。 RYR的结构分析由于其尺寸较大（〜2.3 MDA），位于膜环境和动态性质而异常具有挑战性。迄今为止，单个粒子冷冻EM是对这种大型整合膜蛋白结构分析的最可行的方法。然而，由于冰冻膜中存在洗涤剂，对Ryrs的冷冻EM研究面临着另一个障碍。这引发了许多问题，并优化了冰厚度和EM成像，以产生与可靠的3D重建相对鲜明对比的图像。减少的图像对比度是总体上产生膜蛋白高分辨率结构的主要障碍。通过冷冻EM实现RYR1的高分辨率结构，显然需要在冷冻特异性制备和成像条件的方法中取得突破。在该项目中，我们旨在开发一种用于使用新的表面活性剂，两亲聚合物的单个粒子冷冻EM分析的RYR1玻璃化方法，并代替洗涤剂将通道蛋白溶于及其功能形式保持在无水清洁剂溶液中（AIM1）。通过使用这种方法，我们预计在冷冻EM研究中会绕过清洁剂的困难，并在封闭状态下以亚纳米分辨率在封闭状态下实现RYR1的3D重建。然后，我们建议将RYR1通道重建为小的单层囊泡，并使用单个颗粒重建的变体来求解脂质膜中的通道结构（AIM 2）。将在EMAN/EMAN2软件的框架内为该项目开发新的计算工具，以实现超出当前〜1 nm的分辨率的完整RYR1的结构，并在膜环境中建立其3D体系结构。确定的结构将揭示机械信息丰富的蛋白质特征，这将允许对RYR1通道功能的重要见解。一旦建立了最佳方法论，我们预计将RYR1的结构分析扩展到近原子分辨率，并扩展到不同生理相关的功能状态。作为这项研究的一部分，开发的方法将对其他离子通道和近膜蛋白复合物的研究具有广泛的适用性。