Protein Energy Landscapes by NMR and Single Molecules

核磁共振和单分子的蛋白质能量景观

• 批准号: 6784749
• 负责人: Frederick W. Dahlquist
• 金额: $ 23.15万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6784749
• 关键词: Archaea; atomic force microscopy; bacteriophage T4; bioenergetics; conformation; crosslink; hydropathy; lysozyme; mass spectrometry; mathematical model; nanotechnology; nuclear magnetic resonance spectroscopy; physical model; protein folding; protein structure function; recombinant proteins; stop flow technique; thermostability

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.

描述（由申请人提供）：该建议是针对更好地理解蛋白质结构，稳定性和动力学之间关系的。在能源景观术语中，我们建议研究其能源最小值附近的景观，从最小值到展开状态的能源屏障的性质，以及在高度变性条件下看到的结构化状态在产生增加的热稳定性方面的作用。该提案包括3个特定目的：（1）我们建议使用现代的核磁共振方法，尤其是放松分散技术来检测和定义蛋白质的少数构象。这些激发的状态通常可能是配体结合，折叠无折叠途径和其他结构变化很重要的事件的关键中间体。提出了实验来检查核心堆积缺陷，低pH和变性剂对少数平衡物种的性质和分布的作用。 （2）开发了检查单蛋白分子对旨在展开蛋白质的机械力的施用所需的方法。这种方法将通过两个双链DNA“手柄”连接一个单个蛋白质分子，以在两个不同珠的特定位点附加。通过使用激光镊子将珠子拉开，将力施加在蛋白质上。这种方法通过直接测量蛋白质通过将蛋白拉开来扩展蛋白质所需的力来研究蛋白质折叠的能力。这将使我们能够沿特定的反应坐标获得与附着点之间的距离相对应的特定反应坐标的新视角。 （3）了解蛋白质的热稳定性的结构和热力学来源。使用Thermotoga Maritima的Chey蛋白作为模型，我们发现其热稳定性在很大程度上是由于其折叠时的热容量变化异常较低。这种不寻常的热容量变化似乎是高度结构化的状态的结果，并提出了实验来研究热容量变化和展开状态性质的结构碱基。