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; 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）：该项目的目的是了解酶如何调整其能量景观以执行其功能。众所周知，蛋白质实际上是构象状态的合奏，而不是通常用来描绘它们的静态结构。作为功​​能的一部分，蛋白质经历结构和动态变化的事实表明，合奏对于其功能至关重要。这一发现的含义是，除了编码三维结构外，蛋白质的序列还演变为为整个能量景观（即构象合奏）的编码。知道这是如何做到的，这是非常重要的。是否有将蛋白质与不同功能联系起来的统一原则？在这里，我们利用我们小组在上一个赠款期间的独特发现，这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促活性 - 本质上，能量景观在其功能上重要的库存中展开。这种构象变化模式与当前接受的模型形成鲜明对比，因此，据信开口和闭合反应促进催化转移。在该提案中，我们利用这种不断发展的反应为一种突变策略，旨在研究AK不同区域之间的耦合，以及该耦合如何产生构成AK能量景观的国家集合。我们的方法旨在了解构象波动在功能中的作用，并核对观察结果，即1）在没有结构变化的情况下，蛋白质的动态变化可能发生； 2）功能可以与蛋白质不同区域的稳定性相关； 3）结合可以在许多站点增加（而不是减少）动力学； 4）在没有结构或动态变化的途径的情况下，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（CD）进行结合和稳定性测量，监测热展开和氢交换（HX），我们将使用NMR 15N放松分散（CPMG）和稳态分析监测构象和酶促过程的动力学。我们的研究设计为在AK的能源景观中阐明了国家，并了解它们在推动催化过程中所起的作用。