Development and Application of Methods for Investigating the Biological Roles and Structural Basis of Protein Kinetic Stability

研究蛋白质动力学稳定性的生物学作用和结构基础的方法的开发和应用

• 批准号: 1158375
• 负责人: Wilfredo Colon
• 金额: $ 119.47万
• 依托单位: Rensselaer Polytechnic Institute
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1158375&HistoricalAwards=false
• 关键词: Development; Application; Methods; Investigating; Biological

Intellectual MeritLike a child strapped to a high-chair, some proteins are trapped in their native structure, unable to access partially and globally unfolded conformations. This may be a method used by "mother nature" to protect certain proteins from premature degradation, which would compromise the well being of the organism. Proteins that possess this property are described as being kinetically stable because, unlike most proteins, their long "shelf-life" and stability towards degradation is kinetically controlled by its very slow unfolding rate. The structural basis and biological significance of protein kinetic stability remains poorly understood. Therefore, the main goal of this project is to gain novel insight into the role of kinetically stable proteins (KSPs) in the survival and adaptation of prokaryotic thermophiles and mesophiles, and to gain enhanced understanding about the structural basis of kinetic stability. The central dogma is that bacteria possess KSPs with functions that are essential to survive in their respective environments. Novel electrophoresis methods developed in the laboratory of the Principle Investigator will be further developed and applied to: 1) characterize the proteome of KSPs in several thermophilic and mesophilic bacteria; 2) quantify the kinetic stability of hyperstable proteins directly in the cellular lysate of bacteria; and 3) construct an accessible online database of KSPs to explore the structural and functional basis of kinetic stability. It is expected that the results of this project will enhance the understanding of the biological significance of kinetic stability, and will provide a convenient and accessible assay to quantify the kinetic stability of proteins in their biological milieu. Furthermore, it will yield a wealth of accessible data that will stimulate studies to understand the biological roles of KSPs in the survival, adaptation, and evolution of thermophilic and mesophilic organisms. Broader ImpactAs a broader impact, this project will lead to the discovery of KSPs with potential for biotechnology applications of benefit to society. In terms of education, this project will involve postdoctoral, graduate, and undergraduate researchers working together in a stimulating interdisciplinary environment that integrates training and learning. The Principal Investigator is developing a local program (RISE: Research Internship for Community College Student Engagement) for local community college students that show interest in STEM disciplines and have the academic potential and desire to transfer to a four-year university. A RISE student will participate every summer in the project and will receive mentoring and guidance throughout the process of transfer to RPI and for undergraduate studies.This project is jointly supported by the Biomolecular Dynamics, Structure and Function Cluster in the Division of Molecular and Cellular Biosciences, the Physics of Living Systems in the Physics Division and the Chemistry of Life Processes program in the Chemistry Division.

智力优势就像绑在高脚椅上的孩子一样，一些蛋白质被困在其天然结构中，无法获得部分和整体展开的构象。这可能是“大自然”用来保护某些蛋白质免于过早降解的方法，这会损害生物体的健康。具有这种特性的蛋白质被描述为动力学稳定的，因为与大多数蛋白质不同，它们的长“保质期”和降解稳定性是由其非常慢的解折叠速率在动力学上控制的。蛋白质动力学稳定性的结构基础和生物学意义仍然知之甚少。因此，该项目的主要目标是对动力学稳定蛋白（KSP）在原核嗜热菌和嗜温菌的生存和适应中的作用获得新的见解，并加深对动力学稳定性的结构基础的理解。中心法则是细菌拥有 KSP，这些 KSP 具有在各自环境中生存所必需的功能。原理研究员实验室开发的新型电泳方法将进一步开发并应用于：1）表征几种嗜热和嗜温细菌中KSP的蛋白质组； 2) 直接量化细菌细胞裂解物中超稳定蛋白的动力学稳定性； 3) 构建一个可访问的 KSP 在线数据库，以探索动力学稳定性的结构和功能基础。预计该项目的结果将增强对动力学稳定性生物学意义的理解，并将提供一种方便且易于使用的测定方法来量化蛋白质在其生物环境中的动力学稳定性。此外，它将产生大量可获取的数据，从而促进研究了解 KSP 在嗜热和中温生物体的生存、适应和进化中的生物学作用。更广泛的影响 作为更广泛的影响，该项目将导致发现具有造福社会的生物技术应用潜力的 KSP。在教育方面，该项目将涉及博士后、研究生和本科生研究人员在一个集培训和学习于一体的刺激的跨学科环境中共同工作。首席研究员正在为对 STEM 学科表现出兴趣、具有学术潜力并希望转入四年制大学的当地社区学院学生制定一项本地计划（RISE：社区学院学生参与研究实习）。每年夏天都会有一名RISE学生参加该项目，并在转入RPI和本科学习的整个过程中接受辅导和指导。该项目由分子与细胞生物科学部生物分子动力学、结构与功能集群联合支持，物理系的生命系统物理学和化学系的生命过程化学项目。