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。在能源景观方面，我们建议调查的景观附近的能量最小值，从最小到未折叠状态的能量障碍的性质，和结构化的状态下看到的高度变性的条件下产生的热稳定性增加的可能作用。本研究的主要目的有三：（1）利用现代核磁共振技术，特别是弛豫色散技术来检测和确定蛋白质的少数构象。这些激发态通常是配体结合、折叠-解折叠途径和其他结构变化重要的事件中的关键中间体。实验提出了检查的核心包装缺陷，低pH值和变性剂的性质和少数平衡物种的分布的作用。(2)开发所需的方法来检查单个蛋白质分子对旨在展开蛋白质的机械力的应用的反应。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珠子上的特定位点。通过使用激光镊子将珠子拉开来对蛋白质施加力。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蛋白质所需的力。这将使我们能够获得一个新的视角的能量表面沿着一个特定的反应坐标对应的连接点之间的距离。(3)了解来自超嗜热生物的蛋白质的热稳定性的结构和热力学来源。使用来自海栖热袍菌的CheY蛋白作为模型，我们发现其热稳定性主要是由于其在折叠时热容的异常低的变化。这种不寻常的热容量变化似乎是一个高度结构化的展开状态的结果，并提出实验来研究热容量变化的结构基础和展开状态的性质。

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.