The Experimental Energy Landscape and Protein Function

实验能量景观和蛋白质功能

• 批准号: 10264158
• 负责人: VINCENT J. HILSER
• 金额: $ 42.5万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10264158
• 关键词: Address; Adopted; Affinity; Binding; Binding Sites; Calorimetry; Catalysis; Circular Dichroism; Complex; Databases; Development; Disease; Entropy; Enzymes; Equilibrium; Escherichia coli; Exhibits; Expeditions; Fishes; Funding; Glycine; Goals; Grant; Hydrogen; Kinetics; Knowledge; Learning; Ligands; Measurement; Measures; Mediating; Modeling; Molecular; Molecular Conformation; Monitor; Motion; Movement; Mutation; Nature; Pathway interactions; Play; Positioning Attribute; Predisposition; Probability; Process; Protein Engineering; Proteins; Publishing; Reaction; Regulation; Resolution; Role; Signal Transduction; Structural Protein; Structure; Surface; System; Testing; Therapeutic; Thermodynamics; Titrations; adenylate kinase; base; design; enzyme mechanism; experimental study; improved functioning; insight; mutant; predictive modeling; protein data bank; protein function; structural biology

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.

项目概要： 这个项目的目标是了解酶如何利用构象波动，特别是局部 展开，以促进催化。在过去的几十年里，越来越清楚的是， 作为静态结构存在的蛋白质实际上是有时非常不同的构象状态的集合体， 而波动对功能至关重要。知道这是如何做到的是很重要的。是否有统一的 将不同功能的蛋白质联系起来的原理？这里我们利用几个关键的发现 我们小组在以前的资助周期中发现，这表明腺苷酸酶 激酶（AK）。大肠杆菌，利用局部解折叠来调节其酶活性--实际上， 在其重要的功能范围内展开。我们发现在LID和 AMPbd结构域和在不同区域的解折叠选择性地调节不同的关键酶， 参数，其中一个盖子的变化调制Km，并且另一个盖子的变化调制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。我们的发现与目前公认的模型（假设 由铰链促进的刚性体打开和关闭反应，该铰链被认为促进催化翻转）。的 事实上，AK是蛋白质数据库（PDB）中3,000多个高分辨率结构的代表， 已经假设（但从未实际证明）利用刚体打开/关闭运动， 促进催化，表明有序/无序波动可能比以前认为的更普遍。 如何普遍展开，以及它的存在如何影响40多年来基于结构生物学的 功能研究？我们的做法是双重的。首先，由于我们的结果直接破坏了AK的现有模型， (and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。 酶的功能。其次，我们必须询问酶的数据库，以确定如何一般 局部的展开和无序。所有酶都利用解折叠吗？我们能不能开发一个定量的， AK和其他酶的实验衍生模型？我们将执行绑定和稳定性测量 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），我们将使用NMR监测构象和酶促过程的动力学 CEST（构象交换饱和转移）和稳态酶分析。