Single molecule studies of protein folding/refolding

描述(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纵技术研究展开-再折叠过程的新方法来补充传统的整体实验方法，从而深入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。蛋白质的不正确折叠可能导致聚集和纤维形成，这也是众所周知的，这会导致许多人类疾病，如囊性纤维化、普里恩和阿尔茨海默氏病。对折叠过程的深入了解也将极大地提高我们基于重组蛋白质设计和制造新材料的能力，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令人好奇的课题，而且在医学上也是一个具有巨大实用价值的领域。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。整体测量不能完全解决这种异质性的问题。替代的折叠路径只能间接推断，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，即一次只研究一个蛋白质分子，将在我们寻求对蛋白质折叠问题的完整理解方面发挥重要的补充作用。该项目的重点将是描述具有不同折叠的三种蛋白质的能量图景。该项目的具体目标是：(1)为AFM/单分子机械手设计和制造一个温度控制器，对单个蛋白质分子的机械展开进行随温度变化的测量。(2)在依赖温度的机械展开实验中测量蛋白质能量谱的粗糙度。测量将在恒定力模式和恒定加载速率模式下进行。(3)用机械f值分析方法表征了三种模型蛋白质的过渡态。将为这些实验合成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去折叠的影响。不同的挤压剂将被用来阐明拥挤对去折叠/复性速率和复性产率的影响。

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.