The Experimental Energy Landscape and Protein Function

项目总结： 这个项目的目标是了解酶是如何利用构象波动的，特别是局部的。 展开，以促进催化作用。在过去的几十年里，人们越来越清楚地看到， 蛋白质以静态结构存在，实际上是有时非常不同的构象状态的集合， 而波动是发挥作用的关键。知道这是如何做到的是非常重要的。有没有统一的 连接具有不同功能的蛋白质的原理？在这里，我们利用了几个关键发现 我们小组在之前的资金周期中的发现，这表明腺苷酸酶 来自大肠杆菌的激酶(AK)，使用局部去折叠来调节其酶活性--实际上就是能量格局 在其重要的功能剧目中展开。我们发现在盖子和盖子上都有展开 AMPbd结构域及其在不同区域的展开选择性地调节不同的关键酶 参数，其中一个盖子改变调制Km，而另一个盖子改变调制Kcat。没想到我们 发现局部展开实际上控制了酶的冷适应，从而直接证明了 功能重要性。我们的发现与目前被接受的模型形成了鲜明对比(该模型假设 刚体打开和关闭反应，由铰链促进，据信有助于催化周转)。这个 AK代表蛋白质数据库(PDB)中的3,000多个高分辨率结构) 已经假设(但从未实际演示)利用刚体打开/关闭运动来 促进催化，表明有序/无序波动可能比之前认为的更普遍。 它是如何展开的，它的存在如何影响了40多年来以结构生物学为基础的 功能研究？我们的方法是两方面的。首先，由于我们的结果直接破坏了AK的现有模型 (因此需要一个新的模型)我们将确定我们发现的无序状态是如何负责的 对于酶的功能。其次，我们必须询问酶的数据库以确定有多普遍 局部的展开和无序是。所有的酶都利用去折叠吗？我们能不能开发一种量化的， AK和其他酶的实验模型？我们将进行约束性和稳定性测量 用等温滴定量热法(ITC)、圆二色谱(CD)监测热展开和氢 交换(HX)，我们将使用核磁共振监测构象和酶过程的动力学 CEST(构象交换饱和转移)和稳态酶分析。

Project Summary: The goal of this project is to understand how enzymes utilize conformational fluctuations in particular, local unfolding, to facilitate catalysis. Over the past several 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 connect proteins with different functions? Here we take advantage of several key discoveries discoveries by our group during the 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. We found unfolding to occur in both the LID and the AMPbd domains and that unfolding in the different regions selectively modulated different key enzymatic parameters, with changes in one lid modulating Km , and changes in the other modulating kcat. Unexpectedly we found that local unfolding actually controlled cold adaptation in the enzyme, thus directly demonstrating the functional importance. Our discovery stands in stark contrast to the current accepted model (which posits a rigid-body opening and closing reaction facilitated by a hinge that is believed to facilitate catalytic turnover). The fact that AK is representative of more than 3,000 high-resolution structures in the Protein Data Bank )PDB) that have been hypothesized (but never actually demonstrated) to utilize the rigid-body open/closing motions to facilitate catalysis, suggests that order/disorder fluctuations may be more prevalent than previously believed. How general is unfolding and how does its presence impact the more than 40 years of structural biology-based functional studies? Our approach is two-fold. First, as our results directly undermine the existing models of AK (and thus require a new model) we will determine how the disordered states discovered by us are responsible for the function of the enzyme. Second, we must interrogate the database of enzymes to determine how general local unfolding and disorder are. Do all enzymes utilize unfolding? Can we develop a quantitative, experimentally-derived model of AK and other enzymes? 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 CEST (conformational exchange saturation transfer) and steady state enzymatic analysis.