The experimental energy landscape and protein function

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执行其功能。众所周知，蛋白质实际上是构象状态的集合体，而不是通常用来描述它们的静态结构。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，该集合对其功能至关重要。这一发现的含义是，除了编码三维结构外，蛋白质的序列还进化为编码整个能量景观（即构象集合）。知道如何做这件事是很重要的。有统一的原则将不同功能的蛋白质联系起来吗？在这里，我们利用了我们小组在上一个资助期间的一个独特发现，该发现表明来自大肠杆菌的腺苷酸激酶（AK）使用局部展开来调节其酶活性-本质上，能量景观在其功能重要的库中展开。这种构象变化的模式与目前公认的模式形成鲜明对比，即打开和关闭反应被认为有助于催化转换。在该提案中，我们利用这种展开反应作为突变策略，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能量景观的状态集合。我们的方法是为了理解构象波动在功能中的作用，以及调和以下观察结果：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；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）进行结合和稳定性测量，圆二色性（CD）监测热展开和氢交换（HX），我们将使用核磁共振15N弛散（CPMG）和稳态酶分析来监测构象和酶促过程的动力学。我们的研究旨在阐明AK的能量景观状态，并了解它们在驱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. PUBLIC HEALTH RELEVANCE: The relevance of the proposed studies to understand the structural and dynamic basis of catalysis and function is not just academic. Development of a model that can quantitatively account for the changes in dynamics, thermodynamics and structure would represent the cornerstone of strategies targeted to the design of proteins with new or improved functions or to the design of therapeutic ligands that target those proteins. The studies proposed here are designed to fill the gap in knowledge about how conformationally heterogeneous ensembles can be tuned by Nature to facilitate function.