Folding and Stability of TIM Barrel Proteins
Folding and Stability of TIM Barrel Proteins
批准号:
8632260
负责人:
Osman Bilsel
金额:
$38.12万
依托单位国家:
美国
项目类别:
财政年份:
1976
资助国家:
美国
项目状态:
已结题
起止时间:
1976-05-01 至 2017-12-31
关键词:
Adverse effectsAmino Acid SequenceAmino AcidsBioinformaticsBiological AssayBiological ProcessBiologyBiophysicsBiotechnologyBurialCerealsCollaborationsCollectionComplementComplexComputer SimulationDNA SequenceData AnalysesDependenceDeuteriumElementsEnergy TransferEnzymesEventFluorescence Resonance Energy TransferFree EnergyGenesGlycerophosphatesGoldGrantGrowthHydration statusHydrocarbonsHydrogenHydrogen BondingIn VitroIndiumIndole-3-Glycerol-Phosphate SynthaseIndolesIsoleucineKnock-outLabelLeucineLiteratureLocationMapsMass Spectrum AnalysisMeasuresMedicineMethodsMichiganMolecularMolecular ConformationMutationOntarioOrthologous GeneOutputPathway interactionsPatternPeptidesPlayPopulationProcessPropertyProtein ConformationProtein EngineeringProteinsPulse RadiolysisReactionRelative (related person)RoleSideSiteSolventsSourceStagingStructureSurfaceSurvivorsSystemTechniquesTertiary Protein StructureTestingTimeTryptophanValidationValineVariantWorkYeastsbasecomputer studiesdesignfitnessglobular proteinhuman diseasein vivoinsightinterestmeltingmillisecondmodels and simulationnovelprotein aggregationpublic health relevanceresearch studysimulationthree dimensional structuretool
中文摘要
项目摘要
从分子水平上理解蛋白质的氨基酸序列
将其快速而有效的折叠定向到其天然的、功能构象保持为杰出的
分子生物物理学中的挑战。尽管计算机模拟成功地折叠了小分子蛋白质和
结构域,氨基酸序列在定义物种结构和稳定性方面的精确作用
填充在折叠自由能表面上的物质仍然难以捉摸。我们假设成簇的分枝
脂族侧链,异亮氨酸、亮氨酸和缬氨酸(ILV)是折叠中间体的稳定核心
以及TIM桶状蛋白的天然状态,这是生物学中最常见的基序之一。我们提出了一个多-
在三个吲哚-3-甘油磷酸合成酶(IGPS)同源基因上对这一假设的分面检验,其低
序列同一性导致其常驻ILV簇的大小和位置不同。一系列技术
将探索填充折叠自由能表面的部分折叠状态的结构,并测试它们的
与这些饱和碳氢化合物簇的关系。CD、FRET和SAXS技术将评估二级
构造并提供在微秒时间范围内开始的成对和全局维度信息,
氢交换质谱学和核磁共振方法将绘制稳定的氢键网络图
在毫秒到秒的时间范围内出现的部分折叠状态,以及在这些状态中的侧链掩埋
同样的物种将用一种新的氧化标记方法进行评估。结果将用于验证
使用以自然为中心的GM模型模拟预测部分折叠状态的结构
定义了这些同源序列的整个折叠反应坐标。在一个激动人心的新冒险中,我们将评估
一组详尽的氨基酸替换在所有8条链和之前的环稳定性中的影响
在缺乏同源基因的酵母菌株的生长竞争试验中,影响每个同源基因相对适合度的因素
固有的IGPS基因。假设适合性提供了体内稳定性的估计,这一假设将得到验证
天然态和中间态的稳定性扰动的体外定量评估
SsIGPS同源基因中相同稳定元件中的~100个定点突变的子集。并行CD和
酶活性分析将测量突变对结构和功能的影响
酵素是健康下降的另一种解释。这些体内和体外测量的结果
稳定性扰动将作为生物信息学分析的输入,旨在提供统计-
对突变的上下文依赖性进行重大评估。突变影响的比较
ILV内部和外部的群集将提供一种公正和稳健的方法来确定其
确定球状蛋白稳定核心的意义。
验证了我们的假设,即分支脂肪族侧链簇在
稳定部分折叠状态并引导Tim Barrel蛋白的折叠,有可能具有非常
对生物学、生物技术和医学产生广泛影响。
英文摘要
Project Summary
A molecular-level understanding of the mechanism by which the amino acid sequence of a protein
directs its rapid and efficient folding to its native, functional conformation remains as one of the outstanding
challenges in molecular biophysics. Although computer simulations are successfully folding small proteins and
domains, the precise role of the amino acid sequence in defining the structures and stabilities of the species
that populate the folding free energy surface remains elusive. We hypothesize that clusters of branched
aliphatic side chains, isoleucines, leucines and valines (ILV), serve as cores of stability in folding intermediates
and the native states of TIM barrel proteins, one of the most common motifs in biology. We propose a multi-
faceted test of this hypothesis on a trio of indole-3-glycerolphosphate synthase (IGPS) orthologs, whose low
sequence identity results in varying sizes and locations of their resident ILV clusters. A battery of techniques
will probe the structures of partially-folded states that populate the folding free energy surfaces and test their
relationship with these saturated hydrocarbon clusters. CD, FRET and SAXS techniques will assess secondary
structure and provide pair-wise and global dimensional information beginning in the microsecond time range,
hydrogen-exchange mass spectrometry and NMR methods will map the stable hydrogen bonding networks in
partially-folded states that appear in the milliseconds-to-seconds time frame, and side chain burial in these
same species will be assessed with a novel oxidative labeling method. The results will be used to validate the
predictions of structure in partially-folded states using native-centric GM-model simulations that are capable of
defining the entire folding reaction coordinate of these orthologs. In an exciting new venture, we will assess
the effects of an exhaustive set of amino acid replacements in all 8 ¿-strand and preceding ¿/¿ loop stability
elements on the relative fitness of each ortholog in a growth competition assay in a yeast strain lacking its
intrinsic IGPS gene. The presumption that fitness provides an in vivo estimate of stability will be validated with
an in vitro quantitative assessment of the perturbation of the stability of native and intermediate states in a
subset of ~100 site-directed mutations in the same stability elements in the SsIGPS ortholog. Parallel CD and
enzymatic activity assays will measure the effects of the mutations on the structure and the function of the
enzyme as an alternative explanation for decrease in fitness. The output of these in vivo and in vitro measures
of stability perturbations will serve as input for a bioinformatics analysis designed to offer a statistically-
significant assessment of the context dependence of the mutations. Comparisons of the effects of mutations
within and external to ILV clusters will provide an unbiased and robust approach towards determining their
significance in defining cores of stability in globular proteins.
Validation of our hypothesis, that clusters of branched aliphatic side chains play crucial roles in
stabilizing partially-folded states and guiding the folding of TIM barrel proteins, has the potential to have a very
broad impact on biology, biotechnology and medicine.
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会议论文
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批准号:10009848
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资助金额:$16.81万
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财政年份:2020
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负责人:Osman Bilsel
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批准号:8361315
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项目类别:
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资助金额:$1.77万
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财政年份:2011
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OFF-PATHWAY MISFOLDED FOLDING INTERMEDIATES OF CHEY AND VARIANTS
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批准号:8168649
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资助金额:$0.54万
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PROBING THE STRUCTURE OF THE FOLDING INTERMEDIATE OF CYTOCHROME C
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批准号:8168637
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项目类别:
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资助金额:$1.08万
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财政年份:2010
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负责人:Osman Bilsel
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依托单位:
STRUCTURAL CHANGES IN HUMAN SOD1 BY POST-TRANLATIONAL MODIFICATION
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批准号:8168648
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项目类别:
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资助金额:$0.54万
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财政年份:2010
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负责人:Osman Bilsel
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依托单位:
FOLDING AND ASSEMBLY OF DIMERIC BETA-BARREL PROTEINS
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批准号:7369137
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项目类别:
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资助金额:$2.97万
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财政年份:2006
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负责人:Osman Bilsel
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依托单位:
PROBING THE DENATURED STATES OF TWO BETA-PROTEINS
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批准号:7182103
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项目类别:
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资助金额:$0.73万
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财政年份:2005
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负责人:Osman Bilsel
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依托单位:
TIME RESOLVED SAXS OF MICROSECOND PROTEIN FOLDING INTERMEDIATES
-
批准号:7182104
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项目类别:
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资助金额:$3.1万
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财政年份:2005
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负责人:Osman Bilsel
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依托单位:
FOLDING AND ASSEMBLY OF DIMERIC BETA-BARREL PROTEINS
-
批准号:7182113
-
项目类别:
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资助金额:$1.67万
-
财政年份:2005
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负责人:Osman Bilsel
-
依托单位:
TIME RESOLVED SAXS OF MICROSECOND PROTEIN FOLDING INTERMEDIATES
-
批准号:6975526
-
项目类别:
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资助金额:$5.8万
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财政年份:2004
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负责人:Osman Bilsel
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依托单位:
DENATURED STATES OF TWO BETA-PROTEINS: HIV STRUCTURE
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批准号:6975525
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项目类别:
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资助金额:$3.13万
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财政年份:2004
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负责人:Osman Bilsel
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依托单位:
FOLDING DYNAMICS OF AN ALPHA BETA BARREL PROTEIN
-
批准号:2172855
-
项目类别:
-
资助金额:$3.12万
-
财政年份:1996
-
负责人:Osman Bilsel
-
依托单位:
Folding and Stability of TIM Barrel Proteins
-
批准号:8984304
-
项目类别:
-
资助金额:$37.26万
-
财政年份:1976
-
负责人:Osman Bilsel
-
依托单位:
Folding and Stability of TIM Barrel Proteins
-
批准号:8788409
-
项目类别:
-
资助金额:$37.73万
-
财政年份:1976
-
负责人:Osman Bilsel
-
依托单位:
Folding and Stability of TIM Barrel Proteins
-
批准号:9194405
-
项目类别:
-
资助金额:$37.26万
-
财政年份:1976
-
负责人:Osman Bilsel
-
依托单位:
海外基金