Single molecule studies of protein folding/refolding

蛋白质折叠/重折叠的单分子研究

• 批准号: 6927981
• 负责人: GUOLIANG YANG
• 金额: $ 15万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6927981
• 关键词: atomic force microscopy; bacteriophage T4; bioenergetics; bioengineering /biomedical engineering; computer program /software; mathematical model; muscle proteins; peptide chemical synthesis; protein folding; protein structure; single cell analysis; thermodynamics; ubiquitin

DESCRIPTION (provided by applicant): The long-term goal of the proposed research project is to gain a thorough understanding of the folding mechanisms of proteins by complementing the traditional bulk experimental methods with the novel approach of investigating the unfolding-refolding processes using the single molecule manipulation technique. The vast majority of proteins depend on the correct folding for their proper biological functions. It is also well established that incorrect folding of proteins can lead to aggregation and fibrillogenesis, which cause a number of human diseases, such as cystic fibrosis, prion and Alzheimer's diseases. A deeper understanding of the folding process will also greatly enhance our ability to design and fabricate new materials based on recombinant proteins, and synthesize novel enzymes with specific functions under unusual conditions. Therefore, the protein folding problem is not only a subject of intellectual curiosity, but also an area of enormous practical importance in medicine. Protein folding is a heterogeneous process in which a large number of microscopic pathways connect the unfolded conformations to the unique native structure. Ensemble measurements cannot fully address the problem of this heterogeneity. Alternative folding pathways can only be indirectly inferred, and certain functionally relevant regions of the energy landscape may not be accessible. Therefore, the single molecule technique, in which the protein molecules are studied one at a time, will have an important and complementary role to play in our search for a complete understanding of the protein folding problem. The focus of the project will be on the characterization of the energy landscape of three proteins with distinct folds. The specific aims of the project are: (1) Design and build a temperature controller for the AFM/single molecule manipulator to make temperature dependent measurements on mechanical unfolding of individual protein molecules. (2) Measure the roughness of the energy landscape of proteins in the temperature dependent mechanical unfolding experiments. Measurements will be carried out in both the constant force mode and the constant loading rate mode. (3) Characterize the transition states of three model proteins using the mechanical f-value analysis approach. Polymers of the wt and mutant proteins will be synthesized for these experiments. (4) Investigate the effects of molecular crowding on the unfolding-folding of proteins at the single molecule level. Different crowding agents will be used to elucidate the effects of crowding on the unfolding/refolding rates and refolding yield.

描述（由申请人提供）：所提议的研究项目的长期目标是通过使用单一蛋白质研究去折叠-重折叠过程的新方法来补充传统的批量实验方法，从而彻底了解蛋白质的折叠机制。分子操纵技术。绝大多数蛋白质的正确生物学功能依赖于正确的折叠。众所周知，蛋白质的错误折叠会导致聚集和纤维形成，从而导致许多人类疾病，例如囊性纤维化、朊病毒和阿尔茨海默病。对折叠过程的更深入了解也将大大增强我们设计和制造基于重组蛋白的新材料的能力，以及在异常条件下合成具有特定功能的新型酶的能力。因此，蛋白质折叠问题不仅是一个好奇的课题，而且在医学上也是一个具有巨大实际意义的领域。蛋白质折叠是一个异质过程，其中大量的微观途径将未折叠的构象与独特的天然结构连接起来。集合测量不能完全解决这种异质性问题。替代折叠路径只能间接推断，并且能源景观中某些功能相关的区域可能无法访问。因此，一次研究一个蛋白质分子的单分子技术将在我们寻求全面了解蛋白质折叠问题的过程中发挥重要的补充作用。该项目的重点将是表征具有不同折叠的三种蛋白质的能量景观。该项目的具体目标是：（1）为AFM/单分子操纵器设计和构建温度控制器，以对单个蛋白质分子的机械展开进行温度依赖性测量。 (2) 测量温度依赖性机械展开实验中蛋白质能量图的粗糙度。测量将在恒力模式和恒加载速率模式下进行。 (3) 使用机械 f 值分析方法表征三种模型蛋白质的过渡态。将为这些实验合成野生型和突变型蛋白质的聚合物。 (4) 在单分子水平上研究分子拥挤对蛋白质去折叠的影响。将使用不同的拥挤剂来阐明拥挤对去折叠/重折叠速率和重折叠产量的影响。