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

就像一个被绑在高脚椅上的孩子一样，一些蛋白质被困在它们的天然结构中，无法进入部分和整体展开的构象。这可能是“大自然母亲”用来保护某些蛋白质免于过早降解的一种方法，因为过早降解会损害生物体的健康。具有这种特性的蛋白质被描述为动态稳定的，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缓慢的展开速度在动力学上控制的。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，本项目的主要目标是对动力学稳定蛋白（KSPs）在原核嗜热生物和中温生物的生存和适应中的作用有新的认识，并对动力学稳定性的结构基础有更深入的了解。核心教条是，细菌拥有的KSPs具有在各自环境中生存所必需的功能。在首席研究员实验室开发的新电泳方法将进一步发展和应用于：1)表征几种嗜热和嗜中温细菌中KSPs的蛋白质组；2)直接量化细菌细胞裂解液中超稳定蛋白的动力学稳定性；3)构建可访问的KSPs在线数据库，探索其动力学稳定性的结构和功能基础。期望本项目的研究结果将加深对动力学稳定性的生物学意义的认识，并将为定量蛋白质在其生物环境中的动力学稳定性提供一种方便和可访问的方法。此外，它将产生丰富的可访问数据，这将刺激研究了解KSPs在嗜热和嗜温生物的生存，适应和进化中的生物学作用。更广泛的影响作为一个更广泛的影响，这个项目将导致发现具有潜在的生物技术应用对社会有益的KSPs。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激励的跨学科环境中共同工作，将训练和学习结合起来。首席研究员正在为对STEM学科感兴趣、有学术潜力并希望转学到四年制大学的当地社区大学生开发一个本地项目（RISE：社区大学生参与研究实习）。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获得指导和指导。本项目由分子与细胞生物科学部的生物分子动力学、结构与功能研究小组、物理部的生命系统物理学和化学部的生命过程化学项目共同支持。

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.