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:10684770
- 负责人:
- 金额:$ 29.6万
- 依托单位:
- 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:
- 关键词:Active SitesAddressAntibioticsAromatic Amino AcidsBacteriaBiochemical PathwayBiochemical ProcessBiochemistryBiologicalBiological ModelsCatalysisComplexComputer AnalysisComputer SimulationCrystallographyCysteineDataData SetDiseaseDistantDrug Metabolic DetoxicationElectrostaticsEngineeringEnvironmentEnzymatic BiochemistryEnzyme KineticsEnzymesEquilibriumGoalsHealthHeterogeneityHomeostasisHomologous GeneHydrolysisImpairmentKineticsKnowledgeLearningLifeLightMapsMetabolismMethodologyMethodsModelingModificationMolecular ConformationMotionMutationNatural ProductsOutcomeOxidation-ReductionPathway interactionsPost-Translational Protein ProcessingPropertyProtein DynamicsProteinsPseudomonas fluorescensRalstoniaReactionResistanceResolutionRoleSamplingSignal TransductionSiteSourceStructureSynchrotronsSystemTechniquesTimeVertebral columnViral CancerWorkX ray diffraction analysisX-Ray Crystallographyanti-cancerbasedesigndimerenzyme modelexperimental studyformamideisocyanidemicrobialmolecular dynamicsmutantnew technologynovel therapeuticspreventprotein functiontime resolved datatime usetoolultra high resolutionx-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
依赖半胱氨酸的酶的催化是许多基本的生化途径所必需的,包括
中枢代谢、氧化还原动态平衡和细胞信号。这些通路中的错位发生在
许多疾病状态和以活性半胱氨酸残基为靶点是开发有效的新方法
新药。所有依赖半胱氨酸的酶在催化过程中都是瞬时修饰的,然而人们知之甚少。
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
用发展的时间分辨系列结晶学方法表征重要的功能非平衡
半胱氨酸依赖的异氰化物水合酶在催化过程中的运动。我是校长
使异氰化物解毒的酶,天然产物具有抗菌、抗病毒和抗癌特性。
我们的初步数据表明,在ICH催化过程中,半胱氨酸的瞬时修饰激活了一种非平衡
可以通过混合注入系列X射线结晶学绘制原子细节图的蛋白质动力学。这个
这项建议的目标是开发和应用催化激活的非平衡运动的新模型
通过分析目前从混合注入序列中获得的前所未有的信息丰富的数据集,ICH
结晶学实验。我们将使用系列结晶学和计算方法来确定
半胱氨酸硫酸酯活性部位的瞬时修饰如何激活涉及整体的蛋白质运动
蛋白质,在ICH二聚体中是不对称的,并负责在两个活性部位的动力学异质性
我是二聚体。我们已经创造了改变ICH平衡构象系综IMPAR的突变
催化作用,并降低ICH保护细菌免受异氰化物侵害的能力。使用系列结晶学和
酶动力学,我们将表征这些突变如何改变脑出血催化过程中的变构运动
防止有效的中间水解。最后,我们推广了一个酶运动模型。
通过确定异常侧链和骨架构象应变的作用来确定构象应变
由远方的ICH同系物催化,使X射线衍射到超高分辨率。组合计算,串口
结晶学和酶动力学,我们将确定在ICH催化过程中构象应变如何演变
以前所未有的细节。总之,我们的工作将阐明半胱氨酸的催化修饰如何改变。
酶中的构象系综和非平衡运动。这项工作也将推动急需
同步加速器系列结晶学方法学的进展
这些新的结构生物学技术。
项目成果
期刊论文数量(6)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Reproducibility of protein x-ray diffuse scattering and potential utility for modeling atomic displacement parameters.
- DOI:10.1063/4.0000087
- 发表时间:2021-07
- 期刊:
- 影响因子:0
- 作者:Su Z;Dasgupta M;Poitevin F;Mathews II;van den Bedem H;Wall ME;Yoon CH;Wilson MA
- 通讯作者:Wilson MA
A molecular device for the redox quality control of GroEL/ES substrates.
用于 GroEL/ES 底物氧化还原质量控制的分子装置。
- DOI:10.1016/j.cell.2023.01.013
- 发表时间:2023
- 期刊:
- 影响因子:64.5
- 作者:Dupuy,Emile;VanderVerren,SanderEgbert;Lin,Jiusheng;Wilson,MarkAlan;Dachsbeck,AlixVincent;Viela,Felipe;Latour,Emmanuelle;Gennaris,Alexandra;Vertommen,Didier;Dufrêne,YvesFrédéric;Iorga,BogdanIuliu;Goemans,CamilleVéronique;Rem
- 通讯作者:Rem
Mapping Enzyme Landscapes by Time-Resolved Crystallography with Synchrotron and X-Ray Free Electron Laser Light.
- DOI:10.1146/annurev-biophys-100421-110959
- 发表时间:2022-05-09
- 期刊:
- 影响因子:12.4
- 作者:
- 通讯作者:
Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.
Cucurbit[7]uril 增强自旋标记蛋白质的距离测量。
- DOI:10.1101/2023.08.22.554361
- 发表时间:2023
- 期刊:
- 影响因子:0
- 作者:Yang,Zhimin;Stein,RichardA;Pink,Maren;Madzelan,Peter;Ngendahimana,Thacien;Rajca,Suchada;Wilson,MarkA;Eaton,SandraS;Eaton,GarethR;Mchaourab,HassaneS;Rajca,Andrzej
- 通讯作者:Rajca,Andrzej
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Mark A. Wilson其他文献
Life in a living substrate: Modular endosymbionts of bryozoan hosts from the Devonian of Spain
活基质中的生命:西班牙泥盆纪苔藓虫宿主的模块化内共生体
- DOI:
10.1016/j.palaeo.2020.109897 - 发表时间:
2020 - 期刊:
- 影响因子:0
- 作者:
J. L. Andrés;C. Sendino;Mark A. Wilson - 通讯作者:
Mark A. Wilson
The Performance Appraisal Milieu: A Multilevel Analysis of Context Effects in Performance Ratings
绩效评估环境:绩效评级中情境效应的多层次分析
- DOI:
10.1007/s10869-016-9437-x - 发表时间:
2016 - 期刊:
- 影响因子:4.8
- 作者:
J. K. Ellington;Mark A. Wilson - 通讯作者:
Mark A. Wilson
Origin and paleoecology of Middle Jurassic hiatus concretions from Poland
波兰中侏罗世间断结核的起源和古生态
- DOI:
10.1007/s10347-010-0244-y - 发表时间:
2011 - 期刊:
- 影响因子:1.8
- 作者:
M. Zatoń;Sylwia Machocka;Mark A. Wilson;L. Marynowski;P. Taylor - 通讯作者:
P. Taylor
Preoperative saline loading improves outcome after elective, noncardiac surgical procedures.
术前生理盐水负荷可改善选择性非心脏外科手术后的结果。
- DOI:
- 发表时间:
1996 - 期刊:
- 影响因子:0
- 作者:
R. Garrison;Mark A. Wilson;P. Matheson;David A. Spain - 通讯作者:
David A. Spain
The Ordovician bioclaustration revolution
奥陶纪生物隔离革命
- DOI:
10.1016/j.geobios.2022.10.007 - 发表时间:
2023 - 期刊:
- 影响因子:1.6
- 作者:
O. Vinn;Mark A. Wilson;A. Ernst;U. Toom - 通讯作者:
U. Toom
Mark A. Wilson的其他文献
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{{ truncateString('Mark A. Wilson', 18)}}的其他基金
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
- 批准号:
10259757 - 财政年份:2020
- 资助金额:
$ 29.6万 - 项目类别:
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
- 批准号:
10099548 - 财政年份:2020
- 资助金额:
$ 29.6万 - 项目类别:
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
- 批准号:
10469510 - 财政年份:2020
- 资助金额:
$ 29.6万 - 项目类别:
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