Fundamental Principles of Protein Folding

蛋白质折叠的基本原理

• 批准号: 1517888
• 负责人: C Robert Matthews
• 金额: $ 118.54万
• 依托单位: University of Massachusetts Medical School
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1517888&HistoricalAwards=false
• 关键词: Fundamental; Principles; Protein; Folding

Proteins perform a variety of biological functions, including muscle contraction and cell division. They are also increasingly used in diverse applications in the biotechnology industry ranging from oil spill cleanup to vaccine delivery. The structure and conformational dynamics (i.e. how their structure changes) of proteins are important properties that govern their function. A critical step in altering or designing proteins with specific novel and desirable properties requires a detailed understanding of how the amino acid sequence of a protein codes for its structural and dynamical properties. This project will test the idea that the patterning and separation of hydrophobic (water repelling) and electrically charged amino acids in the protein plays a significant role in modulating these properties. A variety of spectroscopic techniques (using e.g. light and nuclear magnetic resonance) will be used to determine the changes in structure and stability of these proteins and variants of them that have been specially designed. The experimental studies will be complemented by computer simulations to provide insights at the atomic level into the interplay between hydrophobic and electrical charge interactions. The project aims to substantially advance our understanding of protein folding, one of the most important and complex problems in biology. A new venture into specially designed proteins will test the applicability of principles derived from studies of natural proteins and, it is hoped, will lead to new criteria for the design of proteins with novel and useful properties. This project will involve teaching and training of young scientists, which will be enhanced by active participation in the Protein Folding Consortium. The goal of this project is to test the contributions of local-in-sequence and long-range interactions, both in hydrophobic clusters of branched aliphatic side chains and between charged side chains in natural and designed beta/alpha-repeat proteins, in modulating the energy landscape of the proteins. Available evidence on CheY, a naturally occurring protein, suggests that locally connected clusters of isoleucine, leucine and valine side chains rapidly collapse via subdomains that can enhance or impede subsequent folding reactions leading to the native conformation. Recent work on Di-III_14, a de novo designed beta/alpha-repeat protein, suggests that long range electrostatic interactions with high charge segregation are responsible for the formation of very structured intermediates that interconvert extremely slowly with each other and with the native state. The project will extend current knowledge of CheY and Di-III_14 proteins with a battery of spectroscopic methods, at equilibrium and interfaced to ultra-rapid mixing systems, to probe the size, shape and pair-wise distances in the chemically denatured state and in partially folded states that appear in the microsecond time range after dilution to native-favoring conditions. Native-state hydrogen exchange experiments will explore the relationships between partially-folded states and sequence in Di-III_14 and other designed constructs. Mutational analysis will test the role of local and nonlocal ILV clusters and specific electrostatic interactions in the structures formed in high energy states of these proteins, and collaborative single molecule pulling experiments will be used to probe the unique energy surface of Di-III_14 in water. The experimental data will be used to validate collaborative high-resolution molecular dynamics simulations of the folding reactions of CheY and Di-III_14, and new design efforts by collaborators will test the role of charge and charge segregation in molding the free energy surface of beta/alpha-repeat proteins. It is anticipated that the combined application of experimental, computational and design methods to the same targets will substantially enhance our understanding of how sequence determines folding and stability in one of the most common folds in proteins. This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate of Mathematical and Physical Sciences.

蛋白质执行各种生物功能，包括肌肉收缩和细胞分裂。 它们也越来越多地用于生物技术行业的各种应用，从漏油清理到疫苗输送。 蛋白质的结构和构象动力学（即其结构如何变化）是决定其功能的重要特性。 改变或设计具有特定新颖和理想特性的蛋白质的关键步骤需要详细了解蛋白质的氨基酸序列如何编码其结构和动力学特性。 该项目将测试蛋白质中疏水性（斥水）和带电氨基酸的模式化和分离在调节这些特性中起着重要作用的想法。 将使用各种光谱技术（例如使用光和核磁共振）来确定这些蛋白质及其特别设计的变体的结构和稳定性的变化。 实验研究将通过计算机模拟来补充，以在原子水平上深入了解疏水和电荷相互作用之间的相互作用。 该项目旨在大幅推进我们对蛋白质折叠的理解，这是生物学中最重要和最复杂的问题之一。 专门设计的蛋白质的新冒险将测试来自天然蛋白质研究的原则的适用性，并希望将导致新的标准，设计具有新的和有用的特性的蛋白质。该项目将涉及年轻科学家的教学和培训，这将通过积极参与蛋白质折叠联盟而得到加强。该项目的目标是测试本地序列和远程相互作用的贡献，无论是在疏水簇的分支脂肪族侧链和带电侧链之间的天然和设计的β/α-重复蛋白质，在调节蛋白质的能量景观。 关于天然存在的蛋白质CheY的现有证据表明，局部连接的异亮氨酸、亮氨酸和缬氨酸侧链簇通过亚结构域快速折叠，亚结构域可以增强或阻碍随后的折叠反应，导致天然构象。 Di-III_14是一种从头设计的β/α-重复蛋白，最近的研究表明，具有高电荷分离的长程静电相互作用是形成非常结构化的中间体的原因，这些中间体彼此之间以及与天然状态之间的相互转化非常缓慢。 该项目将利用一系列光谱方法扩展CheY和Di-III_14蛋白质的现有知识，在平衡状态下并与超快速混合系统连接，以探测化学变性状态和部分折叠状态下的大小，形状和成对距离，这些状态在稀释到天然有利条件后的微秒时间范围内出现。 天然态氢交换实验将探索Di-III_14和其他设计的构建体中部分折叠状态和序列之间的关系。突变分析将测试局部和非局部ILV簇和特定的静电相互作用在这些蛋白质的高能态形成的结构中的作用，并且协作单分子拉动实验将用于探测Di-III_14在水中的独特能量表面。实验数据将用于验证CheY和Di-III_14折叠反应的协作高分辨率分子动力学模拟，合作者的新设计工作将测试电荷和电荷分离在塑造β/α自由能表面中的作用重复蛋白质。 预计将实验，计算和设计方法结合应用于相同的目标将大大提高我们对序列如何决定蛋白质中最常见折叠之一的折叠和稳定性的理解。 该项目由生物科学理事会分子和细胞生物科学部的分子生物物理学小组和数学和物理科学理事会化学部的生命过程化学方案共同资助。