Protein Energy Landscapes by NMR and Single Molecules

描述（由申请人提供）：本提案旨在更好地理解蛋白质结构，稳定性和动力学之间的关系。在能量景观方面，我们建议研究其能量最小值附近的景观，从最小值到未展开状态的能量障碍的性质，以及在高度变性条件下看到的结构状态在产生增加的热稳定性中的可能作用。本课题主要有三个方面的目标：(1)提出利用现代核磁共振方法，特别是弛豫色散技术，检测和定义蛋白质的少数构象。这些激发态通常是配体结合、折叠-展开途径和其他重要结构变化事件的关键中间体。实验研究了岩心填充缺陷、低pH和变性剂对少数平衡种的性质和分布的影响。(2)开发所需的方法来检查单个蛋白质分子对机械力应用的反应，旨在展开蛋白质。这种方法将通过两个双链DNA“手柄”将单个蛋白质分子连接到两个不同小珠上的特定位置。通过使用激光镊子将珠子分开，对蛋白质施加力。这种方法提供了研究蛋白质折叠的能力，允许直接测量通过将蛋白质从特定点拉开来展开所需的力。这将使我们能够沿着与附着点之间的距离相对应的特定反应坐标获得能量面的新视角。(3)了解超嗜热生物蛋白质热稳定性的结构和热力学来源。我们使用来自海洋热菌的CheY蛋白作为模型，发现其热稳定性主要是由于其折叠时热容量的异常低变化。这种不寻常的热容变化似乎是高度结构化的未展开态的结果，并提出了实验来研究热容变化的结构基础和未展开态的性质。

DESCRIPTION (provided by applicant): This proposal is directed toward a better understanding of the relationships between protein structure, stability and dynamics. In energy landscape terms we propose to investigate the landscape near its energy minimum, the nature of the energy barriers leading from the minimum to unfolded states, and the possible role of structured states seen under highly denaturing conditions in generating increased thermal stability. The proposal consists of 3 specific aims: (1) We propose to use modern nuclear magnetic resonance methods and especially relaxation dispersion techniques to detect and define minority conformations of proteins. These excited states can often be critical intermediates in ligand binding, folding-unfolding pathways and other events where structural change is important. Experiments are proposed to examine the role of core packing defects, low pH and denaturants on the nature and distribution of minority equilibrium species. (2) Develop the methods needed to examine the responses of single protein molecules to the application of mechanical forces designed to unfold the protein. This approach will attach a single protein molecule via two double-stranded DNA "handles" to specific sites on two different beads. Force is exerted on the protein by pulling the beads apart using laser tweezers. This approach offers the ability to study protein folding by allowing direct measurement of the force needed to unfold a protein by pulling it apart from specific points. This will allow us to obtain a new perspective of the energy surface along a specific reaction coordinate corresponding to the distance between the points of attachment. (3) Understand the structural and the thermodynamic source of the thermal stability of proteins from hyperthermophilic organisms. Using the CheY protein from Thermotoga maritima as a model, we have found that its thermal stability is largely due to its unusually low change in heat capacity upon folding. This unusual heat capacity change seems to be the result of a highly structured unfolded state and experiments are proposed to investigate the structural bases of the heat capacity change and nature of the unfolded state.