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:
- 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 SitesAddressAntibioticsAntiviral AgentsAromatic Amino AcidsBacteriaBiochemical PathwayBiochemical ProcessBiochemistryBiologicalBiological ModelsCatalysisComplexComputer AnalysisComputer SimulationCrystallizationCrystallographyCysteineDataData SetDiseaseDistantDrug Metabolic DetoxicationElectrostaticsEngineeringEnvironmentEnzymatic BiochemistryEnzyme KineticsEnzymesEquilibriumFoundationsGoalsHealthHeterogeneityHomeostasisHomologous GeneHydrolysisImpairmentKineticsKnowledgeLifeMapsMetabolismMethodologyMethodsModelingModificationMolecular ConformationMotionMutationNatural ProductsOutcomeOxidation-ReductionPathway interactionsPost-Translational Protein ProcessingPropertyProtein DynamicsProteinsPseudomonas fluorescensRalstoniaReactionResistanceResolutionRoentgen RaysRoleSamplingSideSignal TransductionSiteStructureSynchrotronsSystemTechniquesTimeVertebral columnWorkX-Ray Crystallographyanti-cancerbasecrystallinitydesigndimerenzyme modelexperimental studyformamideisocyanidemicrobialmolecular dynamicsmutantnew technologynovel therapeuticspreventprotein functiontime usetoolx-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
该提案的目的是开发和应用新的催化活化非平衡运动模型,
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。利用连续晶体学,
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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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
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
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
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:
10684770 - 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:
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:
10469510 - 财政年份:2020
- 资助金额:
$ 29.6万 - 项目类别:
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