The Experimental Energy Landscape and Protein Function

实验能量景观和蛋白质功能

• 批准号: 10450194
• 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/10450194
• 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; 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： 该项目的目标是了解酶如何利用构象波动，特别是局部构象波动 在过去的几十年里，越来越清楚的是， 作为静态结构存在，蛋白质实际上是有时非常不同的构象状态的集合， 波动对于功能至关重要，了解如何实现这一点非常重要。 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助周期中的发现表明，腺苷酸酶 来自大肠杆菌的激酶 (AK)，利用局部展开来调节其酶活性——实际上，能量景观 我们发现 LID 和 LID 中都存在展开。 AMPbd 结构域和在不同区域中展开的结构域选择性地调节不同的关键酶 参数，其中一个盖子调制 Km 发生变化，而另一调制 kcat 发生变化。 发现局部展开实际上控制了酶的冷适应，从而直接证明了 我们的发现与当前公认的模型形成鲜明对比（该模型定位了 由铰链促进的刚体打开和关闭反应被认为有助于催化翻转）。 事实上，AK 代表蛋白质数据库 (PDB) 中 3,000 多个高分辨率结构， 已经进化（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/无序波动可能比之前认为的更为普遍。 其发展情况如何？它的存在如何影响 40 多年来基于结构生物学的研究 功能研究？我们的方法有两个：首先，我们的结果直接破坏了 AK 的现有模型。 （因此需要一个新模型）我们将确定我们发现的无序状态如何负责 其次，我们必须查询酶的数据库以确定其通用性。 局部展开和紊乱是不是所有酶都利用展开？ AK 和其他酶的实验衍生模型？我们将进行结合和稳定性测量 使用等温滴定量热法 (ITC)、圆二色性 (CD) 监测热展开和氢 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。