The experimental energy landscape and protein function

 描述（由申请人提供）：本项目的目的是了解酶如何利用构象波动来促进催化。在过去的30年里， 越来越清楚的是，蛋白质不是以静态结构存在，而是有时非常不同的构象状态的集合体，并且波动对功能至关重要。知道这是如何做到的是很重要的。有没有一个统一的原则将不同功能的蛋白质连接起来？在这里，我们利用了我们小组在以前的资助周期中的两个发现，这表明来自大肠杆菌的腺苷酸激酶（AK）。大肠杆菌，使用局部解折叠来调节其酶活性-实际上，能量景观在其功能重要的剧目中具有解折叠。最初，我们发现解折叠只发生在LID结构域中，并且这种解折叠在生理条件下调制Km。最近，我们发现在分子的其他部分展开，表明在某些区域的局部展开调制Km，在其他区域的局部展开调制kcat，并且展开介导结构域之间的通信。这种构象变化模式与目前公认的模型形成鲜明对比，其中刚体铰链打开和关闭反应被认为有助于催化周转。在该提案中，我们利用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AK在催化营业额过程中发生变化的状态的集合。我们的方法建立在我们以前的结果，并面向发展的定量实验推导模型的AK。我们将使用等温滴定量热法（ITC）、圆二色性（CD）监测的热去折叠和氢交换（HX）进行结合和稳定性测量，我们将使用NMR 15 N弛豫分散（CPMG）和稳态酶分析监测构象和酶促过程的动力学。

 DESCRIPTION (provided by applicant): The objective of this project is to understand how enzymes utilize conformational fluctuations to facilitate catalysis. Over the past 3 decades it has become increasingly clear that rather existing as static structures, proteins are actually ensembles of sometimes very different conformational states, and the fluctuations are critical to function. It is of great import to know how this is done. Are there unifying principles that connec proteins with different functions? Here we take advantage of two discoveries by our group during previous funding cycles, which demonstrates that the enzyme adenylate kinase (AK) from E. coli, uses local unfolding to modulate its enzymatic activity - in effect, the energy landscape has unfolding within its functionally important repertoire. Initially we found unfolding to occur only in the LID domain and that this unfolding modulated Km at physiological conditions. More recently we found unfolding in other parts of the molecule, demonstrating that local unfolding in some regions modulates Km, local unfolding in other regions modulates kcat, and that unfolding mediates communication between domains. This mode of conformational change stands in stark contrast to the current accepted model, whereby a rigid-body hinge 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 of AK that changes during the course of catalytic turnover. Our approach builds on our previous results and is geared toward the development of a quantitative experimentally derived model of AK. 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.