Protein Energy Landscapes by NMR and Single Molecules

描述(由申请人提供)：这项建议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，我们建议调查能源最低值附近的景观，从最低能级到展开状态的能垒的性质，以及在高度变性条件下看到的结构化状态在产生更高热稳定性方面的可能作用。该建议包括三个具体目标：(1)我们建议使用现代核磁共振方法，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-展开途径和其他结构变化很重要的事件中的关键中间体。通过实验考察了岩芯填充缺陷、低pH值和变性剂对少数平衡物种的性质和分布的影响。(2)发展检测单个蛋白质分子对设计用于展开蛋白质的机械力的响应所需的方法。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置。通过使用激光镊子将珠子分开，对蛋白质施加作用力。这种方法提供了研究蛋白质折叠的能力，通过将蛋白质从特定的点上拉出来，直接测量蛋白质展开所需的力。这将允许我们沿着与连接点之间的距离相对应的特定反应坐标获得能量表面的新透视。(3)了解高热生物蛋白质热稳定性的结构和热力学来源。以来自Thermotoga maritima的Chey蛋白为模型，我们发现其热稳定性在很大程度上是由于其折叠时热容变化异常低所致。这种不寻常的热容变化似乎是高度结构的展开状态的结果，并提出了实验来研究热容变化的结构基础和未折叠状态的性质。

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.