The experimental energy landscape and protein function

实验能量景观和蛋白质功能

• 批准号: 8474776
• 负责人: VINCENT J. HILSER
• 金额: $ 37.93万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8474776
• 关键词: Accounting; Address; Affect; Affinity; Amino Acid Sequence; Automobile Driving; Binding; Calorimetry; Cataloging; Catalogs; Catalysis; Circular Dichroism; Code; Complex; Coupling; Development; Distal; Entropy; Enzymes; Escherichia coli; Evolution; Glycine; Grant; Hydrogen; Induced Mutation; Kinetics; Knowledge; Ligands; Link; Measurement; Measures; Modeling; Molecular Conformation; Monitor; Mutate; Mutation; Nature; Pathway interactions; Peptide Sequence Determination; Play; Positioning Attribute; Probability; Process; Property; Protein Region; Proteins; Reaction; Relaxation; Research Design; Role; Signal Transduction; Site; Structure; Surface; Testing; Therapeutic; Thermodynamics; Titrations; adenylate kinase; base; design; enzyme activity; enzyme mechanism; improved functioning; insight; interest; mutant; pressure; protein function; protein structure; three dimensional structure

DESCRIPTION (provided by applicant): The objective of this project is to understand how enzymes tune their energy landscapes so as to perform their functions. It is well known that rather than existing as the static structures usually used to depict them, proteins are actually ensembles of conformational states. The fact that proteins undergo both structural and dynamic changes as part of the function indicates that the ensemble is critical to its function. The implication of this finding is that in addition to coding for the three dimensional structures, a protein's sequence also evolved to code for the entire energy landscape (i.e. the ensemble of conformations). It is of great import to know how this is done. Are there unifying principles that connect proteins with different functions? Here we leverage a unique discovery by our group during the previous grant period, which shows that the enzyme adenylate kinase (AK) from E. coli, uses local unfolding to modulate its enzymatic activity - in essence the energy landscape has unfolding within its functionally important repertoire. This mode of conformational change stands in stark contrast to the current accepted model, whereby an opening and closing reaction is believed to facilitate catalytic turnover. In the proposal, we leverage this unfolding reaction into a mutation strategy designed to investigate the coupling between the different regions of AK, and how that coupling produces an ensemble of states that constitutes the energy landscape of AK. Our approach is geared to understanding the role of conformational fluctuations in function, as well as reconciling the observations that, 1) dynamic changes in proteins can occur in the absence of structural changes; 2) function can be correlated to the stability of different regions of the protein; 3) binding can increase (rather than decrease) dynamics at many sites; and 4) structural and dynamic changes can propagate to distal parts of a protein structure in the absence of a pathway of structural or dynamic changes linking the two sites. We will perform binding and stability measurements using isothermal titration calorimetry (ITC), circular dichroism (CD) monitored thermal unfolding and hydrogen exchange (HX), and we will monitor the kinetics of the conformational and enzymatic processes using NMR 15N relaxation dispersion (CPMG) and steady state enzymatic analysis. Our studies are designed elucidate the states in the energy landscape of AK and to understand the role they play in driving the catalytic process.

描述（由申请人提供）：该项目的目标是了解酶如何调整其能量景观以发挥其功能。众所周知，蛋白质实际上是构象状态的集合，而不是通常用来描述它们的静态结构。蛋白质作为功能的一部分经历结构和动态变化这一事实表明，整体对其功能至关重要。这一发现的含义是，除了编码三维结构之外，蛋白质序列还进化为编码整个能量景观（即构象集合）。了解这是如何完成的非常重要。是否存在将具有不同功能的蛋白质联系起来的统一原则？在这里，我们利用了我们小组在上一个资助期间的独特发现，该发现表明来自大肠杆菌的酶腺苷酸激酶（AK）利用局部展开来调节其酶活性——本质上，能量景观已经在其重要的功能范围内展开剧目。这种构象变化模式与目前公认的模型形成鲜明对比，目前公认的模型认为开合反应有助于催化转换。在该提案中，我们将这种展开反应利用到突变策略中，旨在研究 AK 不同区域之间的耦合，以及这种耦合如何产生构成 AK 能量景观的状态集合。我们的方法旨在了解构象波动在功能中的作用，并协调以下观察结果：1）蛋白质的动态变化可以在没有结构变化的情况下发生； 2) 功能可以与蛋白质不同区域的稳定性相关； 3) 结合可以增加（而不是减少）许多位点的动态； 4) 在没有连接两个位点的结构或动态变化途径的情况下，结构和动态变化可以传播到蛋白质结构的远端部分。我们将使用等温滴定量热法 (ITC)、圆二色性 (CD) 监测热解折叠和氢交换 (HX) 进行结合和稳定性测量，并且我们将使用 NMR 15N 弛豫色散 (CPMG) 监测构象和酶促过程的动力学和稳态酶分析。我们的研究旨在阐明 AK 能量格局中的状态并了解它们在驱动催化过程中所发挥的作用。