The experimental energy landscape and protein function

描述(由申请者提供)：本项目的目标是了解酶如何调节它们的能量环境以执行它们的功能。众所周知，蛋白质不是像通常用来描述它们的静态结构那样存在，而是实际上是构象状态的系综。作为功能的一部分，蛋白质经历了结构和动态变化的事实表明，该集成对其功能至关重要。这一发现的含义是，除了编码三维结构外，蛋白质的序列也进化为编码整个能量景观(即构象的集合)。知道这是如何做到的是非常重要的。有没有将具有不同功能的蛋白质联系起来的统一原则？在这里，我们利用了我们的团队在上一次资助期间的一项独特发现，该发现表明，来自大肠杆菌的腺苷酸激酶(AK)酶使用局部去折叠来调节其酶活性--本质上，能量格局已经在其重要的功能谱系中展开。这种构象变化模式与目前公认的模型形成鲜明对比，在该模型中，打开和关闭反应被认为促进了催化剂的周转。在该提案中，我们将这种展开反应转化为突变策略，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能量图景的状态集合。我们的方法旨在了解构象波动在功能中的作用，并符合以下观察结果：1)在没有结构变化的情况下，蛋白质可以发生动态变化；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)许多位点的动力学；以及4)如果没有连接两个位点的结构或动态变化的途径，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热法(ITC)、圆二色谱(CD)监测的热展开和氢交换(HX)来进行结合和稳定性测量，并将使用核磁共振15N弛豫分散(CPMG)和稳态酶分析来监测构象和酶过程的动力学。我们的研究旨在阐明AK的能量格局中的状态，并了解它们在推动催化过程中所起的作用。

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.