Co-Translational Folding of Metamorphic Proteins: Assessing Structure-Function Transitions of the Mitotic Checkpoint Protein MAD2 on the Human Ribosome Surface and in the Presence of Folding Effectors

变态蛋白的共翻译折叠：评估人核糖体表面和折叠效应器存在下有丝分裂检查点蛋白 MAD2 的结构功能转变

• 批准号: 10598244
• 负责人: Shannon Yan
• 金额: $ 8.32万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-08 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10598244
• 关键词: ATP phosphohydrolase; Address; Adopted; Amino Acid Sequence Databases; Binding; Biochemical; Biochemistry; Biological; Biophysics; Cancerous; Cell Cycle; Cell division; Cell physiology; Cells; Cellular biology; Chromosomal Instability; Chromosome Segregation; Complement; Complex; Data; Development; Disease; Dyes; Ensure; Environment; Equilibrium; Event; Genomic Instability; Goals; Growth; Human; Image; In Vitro; International; Knowledge; Label; Lead; Light; MXI1 gene; Malignant Neoplasms; Maps; Mechanics; Mediating; Medical; Mentorship; Metabolic Diseases; Methods; Mitosis; Mitotic; Mitotic Checkpoint; Molecular; Molecular Biology; Molecular Chaperones; Molecular Conformation; Molecular and Cellular Biology; Mutation; Nature; Normal Cell; Pathway interactions; Pattern; Peptides; Phase; Physiological; Potential Energy; Prevalence; Principal Investigator; Program Development; Property; Protein Biosynthesis; Proteins; Records; Regulation; Reporting; Research; Research Institute; Resolution; Ribosomes; Role; Route; SWI1; Scheme; Signal Transduction; Spatial Distribution; Stimulus; Structure; Surface; System; Techniques; Time; Training Programs; Translations; Variant; Work; career; career development; cell growth; cell growth regulation; cellular imaging; conformer; design; functional restoration; genetic manipulation; grasp; imaging study; in vivo; insight; interdisciplinary approach; invention; laser tweezer; mitotic spindle checkpoint protein MAD2; mutant; nanobodies; operation; polypeptide; protein folding; reconstitution; repaired; response; single molecule; skills; tenure track; theories; translation assay; tumorigenesis

Project Summary/Abstract The goal of Dr. Shannon Yan's career development application is to understand the energetic and mechanistic principles that govern structural and functional switching by proteins critical for cellular regulation. Dr. Yan will begin with studying transitions of human mitotic-arrest deficient 2 protein MAD2 in the context of co-translational protein folding, and ultimately focus on its dynamic fold-switching flux established in vivo by folding effectors and chaperones. As structure dictates the function of a biomolecule, MAD2 is capable of switching folds between an inactive open state and mitotic-arrest closed state to modulate the formation of spindle assembly during mitosis—otherwise leading to detrimental chromosome instability or cancerous development. This proposal will explore the molecular details regarding MAD2 structure-function transitions, aiming to elucidate the underlying principles for the operation of protein switches: 1) Is there a default native fold, or an initial equilibrium condition, when MAD2 is first synthesized? Dr. Yan's invention of the first single-human-ribosome translation assay on optical tweezers will directly resolve in real-time the co-translational folding trajectory for a single MAD2 protein as its nascent polypeptide gradually emerges on the human ribosome surface. 2) Is the co-translational folding pathway, and thus the prevalence of one conformer over the other, influenced by folding effectors dependent on the physiological state of cells? Dr. Yan will examine how translation rates, the presence of folding effectors/chaperons, and other cellular factors that may reshape the folding energy landscape for MAD2 protein switch. Knowledge gained from her single-molecule work in the K99 phase will fuel the subsequent cell imaging studies proposed for the R00 phase: 3) How does MAD2 oscillate between different conformer distributions—both spatially and temporally—in accordance with phases of the cell cycle? The flux of open and closed MAD2 protein switches at various stages during mitosis will be characterized and correlated to other concurrent cellular events. Results obtained during this period will enable Dr. Yan to establish herself in the field of cellular biology and cellular mechanics, in which she aims to lead a research group as a tenure-track principal investigator at an academic research institute. Dr. Yan will apply a multidisciplinary approach combining biophysics, molecular and cellular biology, and single-molecule methods, together with genetic manipulations to formulate a unique research line aimed at resolving cellular dynamics and probing the associated cellular mechanics during cell division. This five-year career development program is tailored to prepare Dr. Yan for an independent scientific career. It will build on Dr. Yan's extensive background in ribosome translation and single-molecule techniques, while expanding her skill sets in molecular biology and biochemistry to investigate folding of metamorphic protein switches. Her training program will be directed by Dr. Carlos Bustamante at UC Berkeley—an internationally recognized leader in protein folding and with extensive records of mentorship. Dr. Tanaka and Dr. Legname will also join in to support Dr. Yan's research, professional growth, and her transition into independence. The long-term objective of Dr. Yan's independent research is to expand our understanding on how cells modulate biological signals to grow and multiply, and to identify the origins of regulatory deviations that lead to diseases in humans.

项目摘要/摘要 Shannon Yan博士的职业发展应用程序的目标是了解充满活力和机械原理 由对细胞调节至关重要的蛋白质进行结构和功能转换。 Yan博士将从 研究在共同翻译蛋白折叠的背景下，人类有丝分裂犯罪2蛋白MAD2的过渡， 并最终专注于通过折叠效应和伴侣在体内建立的动态折叠助剂通量。作为 结构决定生物分子的功能，MAD2能够在不活动的开放状态和 有丝分裂 - 违反状态以调节有丝分裂过程中主轴组件的形成 - 否则导致确定 染色体不稳定性或取消发展。该建议将探讨有关MAD2的分子细节 结构功能过渡，旨在阐明蛋白质开关操作的基本原理：1） 首先合成MAD2时，有默认的本机折叠或初始等效条件吗？ Yan博士的意图 光学镊子上的第一个单人类 - 骨体翻译分析将直接实时解决。 单个MAD2蛋白的折叠轨迹逐渐出现在人类核糖体表面上。 2）是共同翻译途径，因此是一种构象异构体在另一个构象中的患病率，受折叠的影响 效应子取决于细胞的物理状态？ Yan博士将研究翻译率，存在 折叠效果/伴侣和其他可能重塑MAD2蛋白折叠能量景观的细胞因子 转变。在K99阶段，她的单分子工作获得的知识将为随后的细胞成像研究增强 针对R00阶段提出的：3）MAD2如何在不同的构象异构分布之间振荡 - 在空间和 暂时 - 根据细胞周期的阶段？开放和关闭的MAD2蛋白开关的通量在各种 有丝分裂期间的阶段将被表征并与其他并发的细胞事件相关。在此期间获得的结果 时期将使Yan博士能够在细胞生物学和细胞力学领域建立自己的目标，她的目标是 领导研究小组作为学术研究所的终身轨道首席研究员。 Yan博士将申请 结合生物物理学，分子和细胞生物学以及单分子方法的多学科方法，一起 通过遗传操作来制定旨在解决细胞动力学并探测的独特研究线 细胞分裂期间相关的细胞力学。这个五年的职业发展计划是为了准备博士准备的量身定制的。 Yan从事独立的科学生涯。它将建立在Yan博士在核糖体翻译方面的广泛背景和 单分子技术，同时扩展了她在分子生物学和生物化学方面的技能集以研究折叠 变质蛋白开关。她的培训计划将由加州大学伯克利分校的Carlos Bustamante博士指挥 国际公认的蛋白质折叠领导者以及广泛的心态记录。田中博士和博士 Legname还将加入，以支持Yan博士的研究，专业成长以及她向独立的过渡。这 Yan博士独立研究的长期目标是扩展我们对细胞如何调节生物学的理解 信号生长和繁殖，并确定导致人类疾病的监管出发的起源。