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偶联）是真正的生物学功能必不可少的真正的电化学信号转导的示例，并且是通过短暂的，依赖电压依赖性的构象变化来实现二氢吡啶受体（DHPR，DHPR，钙通道）？和 ？肌肉细胞表面上的亚基与肌质网上的ryanodine受体（RYR）相互作用。电压触发DHPR（细胞表面）和RYR（细胞内）通道之间的关键蛋白质蛋白质相互作用的事实代表了其在细胞外研究的重要技术障碍。 DHPR和RYR之间静态和电压驱动的瞬态蛋白质 - 蛋白质相互作用的捕获和表征将需要在肌肉中扩大遗传密码，并同时扩展实验性砷的扩展，以研究肌肉生物学。我们的长期目标是在化学生物学的迅速发展领域中应用进步来测试长期存在的假设：电压驱动的蛋白质 - 蛋白质蛋白（机械）耦合DHPR和RYR之间的偶联促进了内部商店的Ca2+迅速释放。拟议的研究的目的是应用创新的化学生物学方法 - 将遗传编码的合成氨基酸苯甲酮苯甲酮phe（BPA）纳入具有独特的光活化交联活动DHPR的遗传性化学生物学方法 - 在骨骼肌肉的环境中，使用它可以共价触发，否则会诱使其触发型，均无目的蛋白质蛋白质蛋白质蛋白质。几项关键创新使我们的研究可行：使用逆转和腺病毒表达正交tRNA和BPA合成酶基因；涉及以前用于生成Fluo-AM染料的酯缺乏策略的大规模制剂，以产生一种BPA形式，该BPA高度溶于溶解性，无毒和生物合成性，可以使遗传掺入肌肉中；而且，虽然不是我们目前目标的主题，但我们在相关的电压门控钠通道中展示了原则BPA证明，以实现快速（毫秒）的照相链接。这里提出的实验将基于这些进步 - 使用各种肌肉细胞模型，包括肌管培养 - 将这些方法适应DHPR -RYR复合物的分析。在AIM 1中，我们将以A和B接口的现有晶体结构为指导，作为DHPR中遗传编码光化学的模型系统，在AIM 2中，我们将确定DHPR中支持电压驱动相互作用的DHPR的氨基酸侧链。这项研究的贡献将是对肌肉中短暂蛋白相互作用的研究的新工具（技术，试剂，概念），以及对DHPR结构的理解，这对于识别IT与RYR之间的So-Far难以捉摸的分子相互作用至关重要。这些贡献将具有重要意义，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“筹集所有船” 肌肉生物学。