Fundamental Principles of Protein Folding

蛋白质执行多种生物功能，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，从石油泄漏清理到疫苗输送。蛋白质的结构和构象动力学（即它们的结构如何变化）是控制其功能的重要特性。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（疏水）和带电氨基酸的模式和分离在调节这些特性中起重要作用的想法。各种光谱技术（例如光和核磁共振）将被用来确定这些蛋白质的结构和稳定性的变化以及它们的特殊设计变体。实验研究将辅以计算机模拟，以提供在原子水平上对疏水和电荷相互作用之间相互作用的见解。该项目旨在大大提高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。一项针对特殊设计蛋白质的新研究将测试从天然蛋白质研究中得出的原理的适用性，并有望为设计具有新颖和有用特性的蛋白质带来新的标准。该项目将涉及对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。这个项目的目标是测试局部序列和远程相互作用的贡献，无论是在支链脂肪侧链的疏水簇中，还是在天然和设计的β / α重复蛋白的带电荷侧链之间，在调节蛋白质的能量格局方面。关于CheY（一种天然存在的蛋白质）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迅速坍塌，这可以增强或阻碍随后的折叠反应，从而导致天然构象。最近对从头设计的β / α重复蛋白Di-III_14的研究表明，具有高电荷隔离的长距离静电相互作用负责形成非常结构化的中间体，这些中间体彼此之间以及与天然状态的相互转化非常缓慢。该项目将扩展CheY和Di-III_14蛋白的现有知识，使用一系列光谱方法，在平衡状态和超快速混合系统界面，探测化学变性状态和部分折叠状态下的尺寸，形状和成对距离，这些状态在稀释到天然有利条件后出现在微秒时间范围内。原生态氢交换实验将探索Di-III_14和其他设计结构中部分折叠态与序列之间的关系。突变分析将测试局部和非局部ILV簇以及特定静电相互作用在这些蛋白质高能态形成的结构中的作用，协作单分子拉实验将用于探测水中Di-III_14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，合作者的新设计成果将测试电荷和电荷分离在塑造β / α重复蛋白自由能表面中的作用。预计实验、计算和设计方法的结合应用将大大提高我们对蛋白质中最常见折叠之一的序列如何决定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集群和数学与物理科学理事会化学部的生命过程化学项目共同资助。

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.