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解折叠-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。还公认的是，蛋白质的不正确折叠可导致聚集和原纤维形成，这引起许多人类疾病，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，并在不寻常的条件下合成具有特定功能的新型酶。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连接到独特的天然结构。包围测量不能完全解决这种异质性的问题。替代折叠途径只能间接推断，能源景观的某些功能相关区域可能无法访问。因此，单分子技术，其中蛋白质分子的研究，一次一个，将有一个重要的和互补的作用，在我们的搜索，以全面了解蛋白质折叠的问题。该项目的重点将是对具有不同折叠的三种蛋白质的能量景观的表征。本课题的具体目标是：（1）设计并搭建一个用于AFM/单分子操作器的温度控制器，实现对单个蛋白质分子机械解折叠的温度相关测量。(2)在温度依赖性机械去折叠实验中测量蛋白质能量景观的粗糙度。将在恒定力模式和恒定加载速率模式下进行测量。(3)使用机械f值分析方法表征三种模型蛋白质的过渡态。将合成wt和突变蛋白的聚合物用于这些实验。(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/重折叠速率和重折叠产率的影响。

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.