Designer DHPRs, EC coupling and an expanded genetic code in skeletal muscle

骨骼肌中设计的 DHPR、EC 耦合和扩展遗传密码

• 批准号: 8766411
• 负责人: Christopher A Ahern
• 金额: $ 19.24万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8766411
• 关键词: Adenoviruses; Amino Acids; Amino Acyl-tRNA Synthetases; Benzophenones; Binding; Biochemical; Biological; Biological Models; Biological Process; Biology; Calcium Channel; Cardiac; Cell model; Cell surface; Cells; Chemicals; Codon Nucleotides; Communities; Complex; Coupling; Dihydropyridine Receptors; Dyes; Engineering; Environment; Generations; Genes; Genetic; Genetic Code; Goals; Life; Ligase; Literature; Mechanics; Mediating; Membrane; Molecular Conformation; Muscle; Muscle Cells; Muscle Fibers; Phenylalanine; Photochemistry; Preparation; Proteins; Reagent; Research; Retroviridae; RyR1; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcoplasmic Reticulum; Side; Signal Transduction; Site; Skeletal Muscle; Sodium Channel; Structure; System; Techniques; Testing; Transfer RNA; Tyrosine; Work; cell type; crosslink; in vivo; innovation; insight; millisecond; novel; protein complex; protein protein interaction; public health relevance; research study; skeletal; success; technological innovation; tool; tryptophyltyrosine; voltage

DESCRIPTION (provided by applicant): Excitation contraction coupling (EC-coupling) is an example of bona fide electro-chemical signal transduction essential to biological function, and is executed through short-lived, voltage-dependent changes in conformation that enable the dihydropyridine receptor (DHPR, a calcium channel) ? and ? subunits on the muscle-cell surface to interact with ryanodine receptor (RyR) on the sarcoplasmic reticulum. The fact that voltage triggers the key protein-protein interactions between DHPR (on the cell surface) and RyR (intracellular) channels represents a significant technical barrier to their study outside the cell. The capture and characterization of static and voltage-driven transient protein-protein interactions between DHPR and RyR will require expansion of the genetic code in muscle and, concomitantly, expansion of the experimental arsenal for the study of muscle biology. Our long-term goal is to apply advances in the burgeoning field of chemical biology to testing the long-standing hypothesis that voltage-driven protein-protein (mechanical) coupling between DHPR and RyR facilitates rapid release of Ca2+ from the internal stores. The objective of the proposed research is to apply an innovative chemical biology approach - the incorporation of a genetically encoded synthetic amino acid benzophenone-Phe (Bpa) with a unique photoactivated crosslinking activity into designer DHPR's - in the environment of skeletal muscle, using it to covalently trap otherwise elusive, transient protein complexes. Several key innovations make our study feasible: the use of retro- and adenovirus to express the orthogonal tRNA and Bpa synthetase genes; large-scale preparations involving esterfication strategies used previously to generate Fluo-AM dyes to produce a form of Bpa that is highly soluble, non-toxic and biosynthetically competent for genetic incorporation into muscle; and, while not the subject of our present objective, we show proof-of-principle Bpa incorporation into related voltage-gated sodium channels to enable rapid (millisecond-scale) photo-crosslinking. The experiments proposed here will build on these advances - using a variety of muscle-cell models, including myotube cultures - adapting these approaches to the analysis of DHPR-RyR complexes. In Aim 1 we will be guided by an existing crystal structure of the a- and b-interface as a model system for genetically encoded photochemistry in DHPR's, and in Aim 2 we will identify the amino- acid side chains in the DHPR that support voltage-driven interactions with Ryr. The contributions of this study will be new tools (techniques, reagents, concepts) for the study of transient protein interactions in muscle, and an understanding of DHPR structure that will be essential to identifying the so-far elusive molecular interactions between it and the RyR. These contributions will be significant in that our conceptual innovations and technological breakthroughs will provide fresh insight on a previously intractable mechanism, while 'raising all boats' in the field of muscle biology.

描述（由申请人提供）：激发收缩耦合（EC-耦合）是对生物功能至关重要的真正电化学信号转导的一个例子，它是通过短暂的、电压依赖性构象变化来实现的，从而使二氢吡啶受体能够发挥作用（DHPR，钙通道）？和 ？肌细胞表面的亚基与肌浆网上的兰尼碱受体 (RyR) 相互作用。电压触发 DHPR（细胞表面）和 RyR（细胞内）通道之间关键的蛋白质-蛋白质相互作用这一事实，为他们在细胞外的研究带来了重大的技术障碍。 DHPR 和 RyR 之间静态和电压驱动的瞬时蛋白质-蛋白质相互作用的捕获和表征将需要扩展肌肉中的遗传密码，同时还需要扩展用于肌肉生物学研究的实验库。我们的长期目标是应用化学生物学这一新兴领域的进步来检验长期以来的假设，即 DHPR 和 RyR 之间的电压驱动的蛋白质-蛋白质（机械）耦合促进内部储存的 Ca2+ 快速释放。拟议研究的目的是应用一种创新的化学生物学方法——将具有独特光活化交联活性的基因编码合成氨基酸二苯甲酮-Phe (Bpa) 纳入设计者 DHPR 中——在骨骼肌环境中，用它来共价捕获难以捉摸的瞬时蛋白质复合物。几项关键创新使我们的研究变得可行：使用逆转录病毒和腺病毒表达正交 tRNA 和 Bpa 合成酶基因；大规模制备涉及先前用于生成 Fluo-AM 染料的酯化策略，以产生一种高度可溶、无毒且具有生物合成能力的 Bpa，可将基因整合到肌肉中；而且，虽然不是我们当前目标的主题，但我们展示了将 Bpa 纳入相关电压门控钠通道以实现快速（毫秒级）光交联的原理验证。这里提出的实验将建立在这些进展的基础上 - 使用各种肌肉细胞模型，包括肌管培养 - 使这些方法适应 DHPR-RyR 复合物的分析。在目标 1 中，我们将以 a 和 b 界面的现有晶体结构为指导，作为 DHPR 中基因编码光化学的模型系统，在目标 2 中，我们将识别 DHPR 中支持电压的氨基酸侧链- 与 Ryr 驱动的互动。这项研究的贡献将是用于研究肌肉中瞬时蛋白质相互作用的新工具（技术、试剂、概念），以及对 DHPR 结构的理解，这对于识别 DHPR 与 RyR 之间迄今为止难以捉摸的分子相互作用至关重要。这些贡献意义重大，因为我们的概念创新和技术突破将为以前棘手的机制提供新的见解，同时在该领域“举起所有的船” 肌肉生物学。