CAREER: Thermodynamic and Structural Mechanisms in Protein Fold Adaptation and Thermostability

职业：蛋白质折叠适应和热稳定性的热力学和结构机制

• 批准号: 1054787
• 负责人: Nicholas Panasik
• 金额: $ 75万
• 依托单位: Claflin University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1054787&HistoricalAwards=false
• 关键词: CAREER; Thermodynamic; Structural; Mechanisms; Protein

Intellectual MeritUnderstanding how to redesign enzymes to function at different temperatures is critical to increasing the efficiency of many industrial processes including the production of biofuels. It is also crucially important to understanding the basic principles of protein folding, adaptation, and stability. This project tests and applies two novel hypotheses in protein adaptation and stability: 1) that protein adaptation is "Fold-Specific," meaning that the molecular adaptation strategy taken to alter an enzyme's temperature range is dependent on its specific enzymatic fold and 2) that increases in the stability of the native state of a protein can be achieved by limiting the conformations accessible in the unfolded state through steric exclusion of non-native like conformations - a novel mechanism termed 'Entropic Stabilization.' Already, many mutations that have no apparent effect on the folded state structure have been observed to increase thermostability, and the hypotheses stated above are proposed as a fundamental mechanism to explain this phenomenon. In the process of testing these hypotheses, several enzymes (alpha-glucosidases and cellulases) in the most common protein fold, the 8 stranded alpha/beta barrels, will be redesigned to function at the high temperatures consistent with the needs of promising industrial processes. Using high throughput, low error rate, random mutagenesis and chromogenic selection, several enzymes which show increased thermostability will be selected. In order to achieve higher catalytic rates and efficiencies, enzymes from psychrophilic, or "cold-loving," organisms will be used as a starting point. From multiple attempts of this process, a structural map of "hot spots" in the alpha/beta barrel architecture that show a high probability of involvement in temperature adaptation will be generated. These "hot spots" will be compared to those identified from a structural database, developed by this project, of all known alpha/beta barrel structures from mesophilic, thermophilic, and psychrophilic bacteria to posit general, fold specific rules for protein design.In a complimentary approach, those thermostable mutants that exhibit no obvious structural mechanisms for thermostability in the native-state will be compared using hard-sphere steric ab initio molecular modeling to enumerate all possible sterically-allowed conformations for tetra-peptide sequences in the protein. These will be classified as native-like and non-native and will then be compared to similarly calculated conformational space ratios for the structurally equivalent regions of wild-type enzyme and correlated to their propensity for increasing thermostability. In order to classify which conformations are ruled in or out, and which are native-like and non native, a novel 3D Ramachandran Map-like tool that graphically represents different tetrapeptide conformations will be developed. Broader ImpactIn 2009, Forbes ranked Claflin University as the Best Historically Black University in the nation. Over 90% of the student body is African American and 60% are first generation college students, making Claflin a vital part of the regional and national effort to recruit minority students into advanced degrees in STEM fields. By incorporating this research into four different project-based lab courses at both graduate and undergraduate levels, students will learn traditional techniques such as protein purification, PCR, kinetics measurements, and bio computational analysis, and will apply them in semester-long projects. In order to stimulate enthusiasm, the top-performing students (with the largest number of successful mutants or best computational results) will earn one trip to a national research conference to present their results. Thus, this project encourages over 40 African American and first generation students per semester (all biology and biochemistry majors at Claflin) to participate in and learn about cutting edge bio fuel research. The project will introduce the field to a whole generation of students, while at the same time supporting the development of sustainable energy by unlocking a critical step in biofuel production.

了解如何重新设计酶在不同温度下发挥作用，对于提高许多工业过程（包括生物燃料生产）的效率至关重要。这对于理解蛋白质折叠、适应和稳定性的基本原理也至关重要。该项目测试并应用了蛋白质适应性和稳定性的两个新假设：1）蛋白质适应是“折叠特异性的，这意味着改变酶的温度范围所采取的分子适应策略取决于其特定的酶折叠，以及2）蛋白质天然状态稳定性的增加可以通过限制未折叠状态下可接近的构象来实现，排除非天然的类似构象-一种称为“熵稳定”的新机制。“已经观察到许多对折叠状态结构没有明显影响的突变增加了热稳定性，并且上述假设被提出作为解释这种现象的基本机制。在测试这些假设的过程中，最常见的蛋白质折叠中的几种酶（α-葡萄糖苷酶和纤维素酶），即8链α/β桶，将被重新设计，以在高温下发挥作用，符合有前途的工业过程的需求。使用高通量、低错误率、随机诱变和显色选择，将选择显示出增加的热稳定性的几种酶。为了实现更高的催化速率和效率，将使用来自嗜冷或“喜冷”生物的酶作为起点。从这个过程的多次尝试中，将生成α/β桶结构中的“热点”的结构图，其显示参与温度适应的高概率。这些“热点”将与从该项目开发的结构数据库中鉴定的那些进行比较，该数据库包括所有已知的α/β桶结构，从嗜温、嗜热和嗜冷细菌到蛋白质设计的通用折叠特定规则。那些在天然状态下没有表现出明显的热稳定性结构机制的热稳定突变体将使用硬-球位阻从头算分子建模，以枚举蛋白质中四肽序列的所有可能的空间允许构象。这些将被分类为天然样和非天然的，然后将与野生型酶的结构等同区域的类似计算的构象空间比进行比较，并与其增加热稳定性的倾向相关联。为了分类哪些构象被排除或排除，哪些是天然的和非天然的，将开发一种新的3D Ramachandran Map样工具，以图形方式表示不同的四肽构象。更广泛的影响2009年，福布斯将克拉夫林大学评为全国历史上最好的黑人大学。超过90%的学生是非洲裔美国人，60%是第一代大学生，使克拉夫林的区域和国家努力招收少数民族学生进入STEM领域的高级学位的重要组成部分。通过将这项研究纳入四个不同的基于项目的实验室课程在研究生和本科生水平，学生将学习传统的技术，如蛋白质纯化，PCR，动力学测量和生物计算分析，并将它们应用于学期的项目。为了激发热情，表现最好的学生（成功突变体数量最多或计算结果最好）将获得一次参加全国研究会议的机会，以展示他们的成果。因此，该项目鼓励每学期40多名非洲裔美国人和第一代学生（克拉夫林的所有生物学和生物化学专业）参与并了解尖端的生物燃料研究。该项目将向整整一代学生介绍该领域，同时通过解锁生物燃料生产的关键步骤来支持可持续能源的发展。