Biogenesis of human mitochondrial iron-sulfur proteins
人类线粒体铁硫蛋白的生物合成
基本信息
- 批准号:10001537
- 负责人:
- 金额:$ 35.94万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2019
- 资助国家:美国
- 起止时间:2019-09-01 至 2022-08-31
- 项目状态:已结题
- 来源:
- 关键词:Acyl Carrier ProteinAddressAffectAgingApoproteinsBindingBinding SitesBiochemicalBiogenesisBiological AssayCalorimetryCellsChemicalsComplexCysteineDefectDiseaseDissociationEnvironmentEscherichia coliExhibitsExposure toFaceFatty AcidsFerredoxinFluorineGenerationsGrowthHandHeat-Shock Proteins 70HumanIn VitroInvestigationIonsIronIron-Sulfur ProteinsLabelLengthLinkMethodsMitochondriaMolecularMolecular ChaperonesMolecular ConformationMonitorNMR SpectroscopyOrganismPeptidesPlayProcessPropertyProteinsPublishingReactionReportingRoentgen RaysRoleScaffolding ProteinSignal TransductionSiteSourceStructureSulfidesSulfurSulofenurTestingTitrationsX-Ray Crystallographyacyl groupcrosslinkcysteine desulfurasedesignexperimental studyfatty acid metabolismfrataxinhuman diseaseimprovedoverexpressionprotein complex
项目摘要
PROJECT SUMMARY/ABSTRACT
Defects in iron-sulfur cluster assembly or delivery underlie many human diseases and aging processes, yet the
detailed mechanisms for these processes are still unknown. Organisms have evolved machinery consisting of
specialized proteins that operate together to assemble Fe-S clusters efficiently in a way that minimizes cellular
exposure to their toxic constituents (iron and sulfide ions). Many of these proteins are dynamic and participate
in weak complexes that have resisted structural analysis. We are studying these proteins and their interactions
in solution by a combination of NMR spectroscopy, small angle X-ray scattering, chemical crosslinking,
isothermal titration calorimetry, and functional biochemical assays. We are applying this approach to address
important questions regarding the mechanism of assembly of Fe-S clusters in human mitochondria. We are
building on the results of recent X-ray structures that provide a framework for our investigations. Last year, two
independent groups published X-ray structures of the (NFS1-ISD11-Acp)2 complex that exhibited very different
quaternary structures. To determine if these two different structures exist in solution, our approach is to
introduce 19F into NFS1 at a site that is different in the two structures. If two structures are present in solution,
we expect to see separate 19F NMR signals for each. By using 2D NMR experiments, we can investigate the
local environment of the probe and, if separate signals are observed may be able to determine the lifetimes of
each state. To date, all structural and functional studies of the mitochondrial cysteine desulfurase complex
have utilized complexes produced by overexpressing the human proteins NFS1 and ISD11 in E. coli cells. The
resulting (NFS1-ISD11-Acp)2 complex contains the holo-form of E. coli acyl carrier protein (Acp) in place of
human mitochondrial acyl carrier protein (ACP). We will determine structural and functional properties of
(NFS1-ISD11-Acp)2 and (NFS1-ISD11-ACP)2 without an acyl chain and with acyl chains of different lengths to
identify differences between Acp and ACP and to test the published hypothesis that acyl carrier protein in the
cysteine desulfurase complex provides a regulatory link coordinating mitochondrial fatty acid synthesis with
iron sulfur cluster biogenesis. Another aim is to characterize the conformational changes in the cysteine
desulfurase complex that accompany different steps in cluster assembly and the transfer of the assembled
cluster on ISCU to the co-chaperone HSC20. Although it is known that ISCU populates two interconverting
conformations in solution (one structured and one intrinsically disordered), the functional roles of these two
states remain to be elucidated. By separately labeling Trp residues in ISCU and NFS1 with fluorine, we have
determined that 19F NMR enables the observation of different conformational states. These preliminary results
set the stage for experiments designed to answer questions about the roles of the structured and dynamic
states of ISCU and how assembled clusters are transferred from the cysteine desulfurase complex to HSC20.
项目摘要/摘要
铁硫簇组装或交付的缺陷是许多人类疾病和衰老过程的基础
这些过程的详细机制仍然未知。生物已经进化的机器包括
专门的蛋白质一起工作以有效地组装Fe-S簇,以最小化细胞
暴露于其有毒成分(铁和硫化物离子)。这些蛋白质中有许多是动态的,并且参与
在抵抗结构分析的弱复合物中。我们正在研究这些蛋白质及其相互作用
通过NMR光谱,小角度X射线散射,化学交联,在溶液中
等温滴定量热法和功能性生化测定。我们正在应用这种方法来解决
关于人线粒体中Fe-S簇组装机制的重要问题。我们是
基于最近的X射线结构的结果,为我们的调查提供了框架。去年,两个
独立组发表的(NFS1-ISD11-ACP)2复合物的X射线结构非常不同
第四纪结构。为了确定解决方案中是否存在这两个不同的结构,我们的方法是
将19F引入两个结构中不同的站点。如果解决方案中存在两个结构,
我们希望每个人都可以看到每个单独的19F NMR信号。通过使用2D NMR实验,我们可以研究
探针的当地环境,如果观察到单独的信号,可能能够确定
每个状态。迄今为止,线粒体半胱氨酸脱硫酶复合酶复合物的所有结构和功能研究
已经利用了通过过表达大肠杆菌细胞中人类蛋白NFS1和ISD11产生的复合物。这
结果(NFS1-ISD11-ACP)2复合物包含大肠杆菌酰基载体蛋白(ACP)的全面形式
人线粒体酰基载体蛋白(ACP)。我们将确定
(NFS1-ISD11-ACP)2和(NFS1-ISD11-ACP)2,无酰基链,酰基链的长度不同
确定ACP和ACP之间的差异,并测试已发表的假设,即酰基载体蛋白在
半胱氨酸脱硫酶配合物提供了调节连接,将线粒体脂肪酸合成与
铁硫簇生物发生。另一个目的是表征半胱氨酸的构象变化
脱硫酶复合物伴随着集群组件的不同步骤和组装的传递
在ISCU上聚类至副伴侣HSC20。尽管已知ISCU填充了两个互换
溶液中的构象(一种结构化和一个本质上无序),这两个的功能作用
国家仍有待阐明。通过将ISCU和NFS1中的TRP残基与氟标记,我们有
确定19F NMR可以观察到不同的构象状态。这些初步结果
为实验奠定了阶段,以回答有关结构化和动态的作用的问题
ISCU的状态以及聚集的簇如何从半胱氨酸脱硫酶复合物转移到HSC20。
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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JOHN LUTE MARKLEY其他文献
JOHN LUTE MARKLEY的其他文献
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{{ truncateString('JOHN LUTE MARKLEY', 18)}}的其他基金
The BMRB as an evolving resource for biomolecular structure-function research
BMRB 作为生物分子结构功能研究的不断发展的资源
- 批准号:
9462715 - 财政年份:2014
- 资助金额:
$ 35.94万 - 项目类别:
The BMRB as an evolving resource for biomolecular structure-function research
BMRB 作为生物分子结构功能研究的不断发展的资源
- 批准号:
8615052 - 财政年份:2014
- 资助金额:
$ 35.94万 - 项目类别:
The BMRB as an evolving resource for biomolecular structure-function research
BMRB 作为生物分子结构功能研究的不断发展的资源
- 批准号:
9253407 - 财政年份:2014
- 资助金额:
$ 35.94万 - 项目类别:
The BMRB as an evolving resource for biomolecular structure-function research
BMRB 作为生物分子结构功能研究的不断发展的资源
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METABOLITE CHANGES IN E COLI STRAINS EVOLVED TO BE RADIATION RESISTANT
大肠杆菌菌株的代谢物变化进化为抗辐射性
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METHANOCALDOCOCCUS JANNASCHII COBY (MJ1117)
甲烷热球菌 JANNASCHII COBY (MJ1117)
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