Development and Application of Method to Study Protein Kinetic Stability

就像一个被绑在高脚椅上的孩子一样，某些蛋白质被困在它们的天然结构中，无法短暂地对部分和整体展开的构象进行取样。这似乎是“大自然母亲”用来保护某些蛋白质免受降解或错误折叠的一种方法，其中任何一种都会损害蛋白质的功能和生物体的健康。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”和降解稳定性是由缓慢的展开速率在动力学上控制的。这些蛋白质在展开过程中被高能量屏障所困住。这种特性的结构基础和生物学意义尚不清楚。研究人员最近证明了动力学稳定性与蛋白质对洗涤剂十二烷基硫酸钠（SDS）的抗性之间的相关性，并随后开发了一种简单的对角二维（D2D） SDS-聚丙烯酰胺凝胶电泳（SDS- page）方法，用于识别复杂混合物（细胞裂解物）中动力学稳定的蛋白质。本项目旨在将D2D SDS-PAGE方法与质谱和蛋白质组学分析相结合，应用于多种生物，探索动力学稳定性的生物学意义和普遍性，并开发一种量化动力学稳定性的新方法。该项目的结果将有助于更好地理解动力学稳定性的结构基础及其在自然界中的作用。该项目的成果将为主流生物学实验室中蛋白质动力学稳定性的研究提供简单的方法。最后，由于蛋白质越来越多地用于各种应用，包括制药，可再生能源和农业，该项目可能导致延长保质期的蛋白质工程，以造福社会的实际应用。更广泛的影响。该项目将为社区提供一种简单、廉价、敏感和可获得的测定方法，以量化任何感兴趣的蛋白质的动力学稳定性，无论是纯化的还是在其生物环境中。此外，该项目有可能为蛋白质动力学稳定性的生物学作用提供新的见解，可以催化进一步的研究，并推进我们对化学和物理在生命过程中作用的理论理解。从教育的角度来看，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环境中共同工作，将研究训练和学习结合起来。通过实践研究学习是让本科生接触科学及其就业机会的最有效方法。自成立以来，首席研究员的实验室一直致力于培训和指导许多本科生，包括女性和代表性不足的少数民族。本科生是许多出版物的合著者。除了研究训练，参与这个项目的学生将经常参加小组会议，这将提供一个论坛来展示他们的研究和发展他们的沟通技巧。每年参加地方或国家会议将使他们有机会通过海报展示来传播他们的成果。学生将积极参与撰写手稿的早期草稿，以便在同行评审的期刊上发表。

Like a child strapped to a high-chair, certain proteins are trapped in their native structure, unable to transiently sample partially and globally unfolded conformations. This appears to be a method used by "mother nature" to protect some proteins from degradation or misfolding, either one of which would compromise the function of the protein and 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 a slow unfolding rate. These proteins are trapped by a high energy barrier towards unfolding. The structural basis and biological significance of this property remains poorly understood. The investigators recently demonstrated a correlation between kinetic stability and a protein's resistance to the detergent sodium dodecyl sulfate (SDS), and subsequently developed a simple diagonal two-dimensional (D2D) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) method to identify kinetically stable proteins in complex mixtures (cell lysates). The goal of this project is to apply the D2D SDS-PAGE method in combination with mass spectrometry and proteomics analysis to a diverse organisms to explore the biological significance and pervasiveness of kinetic stability, and to develop a new method to quantify kinetic stability. Results of this project should lead to a better understanding of the structural basis of kinetic stability and its roles in nature. The outcome of the project will provide simple methods to facilitate the study of protein kinetic stability in mainstream biology labs. Finally, since proteins are being increasingly used in a variety of applications, including pharmaceuticals, renewable energy, and agriculture, this project could lead to the engineering of proteins with an extended shelf-life for practical applications of benefit to society. Broader impacts. The project will provide the community with a simple, inexpensive, sensitive, and accessible assay to quantify the kinetic stability of any protein of interest, either purified or in its biological milieu. Furthermore, this project has the potential to provide novel insight about the biological role of protein kinetic stability that could catalyze further studies and advance our theoretical understanding about the role of chemistry and physics in life processes. From an educational perspective, this project will involve postdoctoral, graduate, and undergraduate researchers working together in a stimulating environment that integrates research training and learning. Learning through hands-on research is the most effective method for exposing undergraduate students to science and its career opportunities. Since its inception, the lab of the principal investigator has been engaged in training and mentoring many undergraduate students, including women and underrepresented minorities. Undergraduate students have been coauthors in numerous publications. In addition to research training, students involved in this project will participate in frequent group meetings that will provide a forum to present their research and develop their communication skills. Yearly participation at local or national meetings will give them an opportunity to disseminate their results through poster presentations. Students will be actively involved in writing early drafts of manuscripts for publication in peer-reviewed journals.