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.

项目总结/摘要 Shannon Yan博士职业发展申请的目标是了解活力和机械原理 控制着对细胞调节至关重要的蛋白质的结构和功能转换。严医生将开始 在共翻译蛋白质折叠的背景下研究人有丝分裂阻滞缺陷2蛋白MAD 2的转变， 并最终集中于其在体内通过折叠效应子和分子伴侣建立的动态折叠转换通量。作为 结构决定了生物分子的功能，MAD 2能够在非活性开放状态和 有丝分裂停滞闭合状态以调节有丝分裂期间纺锤体组装的形成-否则导致有害的 染色体不稳定或癌变。该提案将探索有关MAD 2的分子细节 结构-功能转换，旨在阐明蛋白质开关操作的基本原理：1） 当MAD 2首次合成时，是否存在默认的天然折叠或初始平衡条件？严博士发明的 第一个在光镊上进行的单人类核糖体翻译测定将直接实时解析共翻译 当单个MAD 2蛋白的新生多肽逐渐出现在人核糖体表面时，其折叠轨迹。 2)是否共翻译折叠途径，以及一种构象异构体相对于另一种构象异构体的普遍性，受到折叠的影响 依赖于细胞生理状态的效应物？严博士将研究如何翻译率，存在的 折叠效应子/伴侣，以及其他可能重塑MAD 2蛋白折叠能量景观的细胞因子 开关.从她在K99阶段的单分子工作中获得的知识将为随后的细胞成像研究提供动力 3）MAD 2如何在不同的构象分布之间振荡-空间上和 根据细胞周期的不同阶段开放和关闭的MAD 2蛋白质开关在不同温度下的通量 有丝分裂期间的阶段将被表征并与其它同时发生的细胞事件相关联。在此期间取得的成果 这段时间将使严博士在细胞生物学和细胞力学领域建立自己的地位，她的目标是 在一家学术研究机构担任终身制首席研究员，领导一个研究小组。严医生会申请 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 通过基因操作，制定了一个独特的研究路线，旨在解决细胞动力学和探索 与细胞分裂过程中的细胞力学相关。这个为期五年的职业发展计划是专为准备博士。 独立的科学生涯。它将建立在严博士在核糖体翻译方面的广泛背景之上， 单分子技术，同时扩大她在分子生物学和生物化学方面的技能，以研究 变态蛋白质开关她的培训计划将由加州大学伯克利分校的卡洛斯布斯塔曼特博士指导。 国际公认的蛋白质折叠领导者，并具有广泛的指导记录。田中博士和 Legname还将加入支持严博士的研究，专业成长和她向独立的过渡。的 严博士独立研究的长期目标是扩大我们对细胞如何调节生物学特性的理解。 信号生长和繁殖，并确定导致人类疾病的监管偏差的起源。