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Time-Resolved X-ray Crystallography of Dynamics in Cysteine-Dependent Enzymes

半胱氨酸依赖性酶动力学的时间分辨 X 射线晶体学

基本信息

批准号：
10099548
负责人：
Mark A. Wilson
金额：
$ 29.6万
依托单位：
UNIVERSITY OF NEBRASKA LINCOLN
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-20 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10099548
关键词：
Active Sites Address Antibiotics Antiviral Agents Aromatic Amino Acids Bacteria Biochemical Pathway Biochemical Process Biochemistry Biological Biological Models Catalysis Complex Computer Analysis Computer Simulation Crystallization Crystallography Cysteine Data Data Set Disease Distant Drug Metabolic Detoxication Electrostatics Engineering Environment Enzymatic Biochemistry Enzyme Kinetics Enzymes Equilibrium Foundations Goals Health Heterogeneity Homeostasis Homologous Gene Hydrolysis Impairment Kinetics Knowledge Life Maps Metabolism Methodology Methods Modeling Modification Molecular Conformation Motion Mutation Natural Products Outcome Oxidation-Reduction Pathway interactions Post-Translational Protein Processing Property Protein Dynamics Proteins Pseudomonas fluorescens Ralstonia Reaction Resistance Resolution Roentgen Rays Role Sampling Side Signal Transduction Site Structure Synchrotrons System Techniques Time Vertebral column Work X-Ray Crystallography anti-cancer base crystallinity design dimer enzyme model experimental study formamide isocyanide microbial molecular dynamics mutant new technology novel therapeutics prevent protein function time use tool x-ray free-electron laser

项目摘要

ABSTRACT Catalysis by cysteine-dependent enzymes is required for many essential biochemical pathways, including central metabolism, redox homeostasis, and cellular signaling. Derangements in these pathways occur in many disease states and targeting reactive cysteine residues is an emerging approach for developing potent new drugs. All cysteine-dependent enzymes are transiently modified during catalysis, however little is known about how cysteine modifications alter the structure and functional dynamics of proteins. We will use newly developed time-resolved serial crystallography methods to characterize functionally important non-equilibrium motions in the cysteine-dependent enzyme isocyanide hydratase (ICH) during catalysis. ICH is the principal enzyme that detoxifies isocyanide natural products that possess antibiotic, antiviral, and anticancer properties. Our preliminary data show that transient cysteine modification during ICH catalysis activates a non-equilibrium protein dynamics that can be mapped in atomic detail by mix-and-inject serial X-ray crystallography. The objective of this proposal is to develop and apply new models of catalysis-activated non-equilibrium motions in ICH by analyzing the unprecedentedly information-rich datasets now available from mix-and-inject serial crystallography experiments. We will use serial crystallography and computational approaches to determine how transient modification of the active site cysteine thiolate activates protein motions that involve the whole protein, are asymmetric in the ICH dimer, and are responsible for kinetic heterogeneity in two active sites of the ICH dimer. We have created mutations that alter the equilibrium ICH conformational ensemble, impair catalysis, and diminish the ability of ICH to protect bacteria from isocyanides. Using serial crystallography and enzyme kinetics, we will characterize how these mutations alter allosteric motions during ICH catalysis and prevent efficient intermediate hydrolysis. Finally, we generalize a model of enzyme motions facilitated by conformational strain by determining the role of unusual side-chain and backbone conformational strain in catalysis by a distant ICH homolog that diffracts X-rays to ultrahigh resolution. Combining computation, serial crystallography, and enzyme kinetics, we will determine how conformational strain evolves during ICH catalysis in unprecedented detail. In total, our work will elucidate how catalytic cysteine modification alters conformational ensembles and non-equilibrium motions in enzymes. This work will also drive urgently needed advances in synchrotron serial crystallography methodology in order to dramatically expand the accessibility of these new structural biological techniques.

摘要半胱氨酸依赖性酶的催化作用是许多基本生化途径所必需的，包括中枢代谢、氧化还原稳态和细胞信号传导。这些通路的紊乱发生在许多疾病状态和靶向反应性半胱氨酸残基是一种新兴的方法，新药所有的半胱氨酸依赖性酶在催化过程中都被瞬时修饰，然而所知甚少半胱氨酸修饰如何改变蛋白质的结构和功能动力学。我们将使用新的开发了时间分辨的系列晶体学方法来表征功能上重要的非平衡态半胱氨酸依赖性酶异氰化物水合酶（ICH）在催化过程中的运动。ICH是主要的使异氰化物天然产物解毒的酶，具有抗生素、抗病毒和抗癌特性。我们的初步数据表明，在ICH催化过程中，短暂的半胱氨酸修饰激活了非平衡蛋白质动力学，可以通过混合和注射系列X射线晶体学绘制原子细节。的本提案的目标是开发和应用催化激活非平衡运动的新模型， ICH通过分析现在可从混合和注入系列中获得的前所未有的信息丰富的数据集，结晶学实验我们将使用连续晶体学和计算方法来确定活性位点巯基化半胱氨酸的瞬时修饰如何激活蛋白质运动，蛋白质，在ICH二聚体中是不对称的，并且负责ICH二聚体的两个活性位点中的动力学异质性。 ICH二聚体。我们已经创造了改变平衡ICH构象整体的突变，催化，并降低ICH保护细菌免受异氰化物侵害的能力。使用连续晶体学和酶动力学，我们将描述这些突变如何改变ICH催化过程中的变构运动，防止有效的中间体水解。最后，我们概括了一个模型的酶运动促进通过确定不寻常的侧链和骨架构象应变的作用，通过远距离ICH同系物的催化作用，衍射X射线至1000分辨率。串行组合计算晶体学和酶动力学，我们将确定在ICH催化过程中构象应变如何演变前所未有的细节。总之，我们的工作将阐明催化半胱氨酸修饰如何改变构象系综和酶的非平衡运动。这项工作还将推动迫切需要的在同步加速器系列晶体学方法的进展，以显着扩大的可及性，这些新的结构生物学技术。