Development and Application of Method to Study Protein Kinetic Stability

蛋白质动力学稳定性研究方法的开发及应用

• 批准号: 0848120
• 负责人: Wilfredo Colon
• 金额: $ 60万
• 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: 2009
• 资助国家: 美国
• 起止时间: 2009-02-15 至 2013-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0848120&HistoricalAwards=false
• 关键词: Development; Application; Method; Study; Protein

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.

就像绑在高脚椅上的孩子一样，某些蛋白质被困在其天然结构中，无法瞬时采样部分和整体展开的构象。这似乎是“大自然”用来保护某些蛋白质免遭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”和降解稳定性是由缓慢的解折叠速率在动力学上控制的。这些蛋白质被高能垒所困，无法展开。这一特性的结构基础和生物学意义仍然知之甚少。研究人员最近证明了动力学稳定性与蛋白质对去垢剂十二烷基硫酸钠 (SDS) 的耐受性之间的相关性，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) 方法来识别动力学稳定性复杂混合物中的蛋白质（细胞裂解物）。该项目的目标是将D2D SDS-PAGE方法结合质谱和蛋白质组学分析应用于多种生物体，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。该项目的结果应该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。该项目的成果将提供简单的方法来促进主流生物学实验室中蛋白质动力学稳定性的研究。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，因此该项目可能会导致蛋白质工程具有更长的保质期，从而实现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、灵敏且易于使用的检测方法，以量化任何感兴趣的蛋白质（无论是纯化的还是在其生物环境中）的动力学稳定性。此外，该项目有可能提供关于蛋白质动力学稳定性的生物学作用的新见解，可以催化进一步的研究并增进我们对化学和物理在生命过程中的作用的理论理解。从教育角度来看，该项目将涉及博士后、研究生和本科生研究人员在一个集研究培训和学习于一体的刺激环境中共同工作。通过实践研究学习是让本科生接触科学及其职业机会的最有效方法。自成立以来，首席研究员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族裔。本科生是许多出版物的合著者。除了研究培训之外，参与该项目的学生还将参加频繁的小组会议，这些会议将提供一个论坛来展示他们的研究并培养他们的沟通技巧。每年参加地方或国家会议将使他们有机会通过海报展示传播其成果。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。