Single molecule studies of protein folding/refolding

描述（由申请人提供）：拟议研究项目的长期目标是通过使用单分子操作技术研究展开-再折叠过程的新方法来补充传统的批量实验方法，从而全面了解蛋白质的折叠机制。绝大多数蛋白质依赖于正确的折叠来实现其适当的生物学功能。此外，不正确的蛋白质折叠可导致聚集和纤维形成，从而导致许多人类疾病，如囊性纤维化、朊病毒和阿尔茨海默病。对折叠过程的深入了解也将大大提高我们设计和制造基于重组蛋白的新材料的能力，以及在特殊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求知欲的主题，而且在医学上也是一个具有巨大实际重要性的领域。蛋白质折叠是一个异质性的过程，在这个过程中，大量的微观途径将未折叠的构象连接到独特的天然结构。集合测量不能完全解决这种异质性的问题。可选择的折叠路径只能间接推断，并且能源景观的某些功能相关区域可能无法进入。因此，单分子技术，其中蛋白质分子一次研究一个，将在我们寻求蛋白质折叠问题的完整理解中发挥重要的补充作用。该项目的重点将是表征三种具有不同褶皱的蛋白质的能量景观。该项目的具体目标是：(1)为AFM/单分子操纵器设计并构建温度控制器，以对单个蛋白质分子的机械展开进行温度依赖测量。(2)测量温度相关力学展开实验中蛋白质能量景观的粗糙度。测量将在恒力模式和恒加载速率模式下进行。(3)利用机械f值分析方法表征三种模型蛋白的过渡态。这些实验将合成wt和突变蛋白的聚合物。(4)在单分子水平上研究分子拥挤对蛋白质展开折叠的影响。将使用不同的拥挤剂来阐明拥挤对展开/再折叠速率和再折叠收率的影响。

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.