STRUCTURAL ANALYSIS OF PROTEIN FOLDING INTERMEDIATES

蛋白质折叠中间体的结构分析

基本信息

  • 批准号:
    6138494
  • 负责人:
  • 金额:
    $ 24.28万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    1997
  • 资助国家:
    美国
  • 起止时间:
    1997-01-01 至 2003-12-31
  • 项目状态:
    已结题

项目摘要

While may small proteins can refold spontaneously in vitro, the identification of the folding pathway, and the detection and structural characterization of folding intermediates have been difficult. Recently, attention has turned to the molten globule state and other nonnative equilibrium states of proteins which are thought to be models for kinetic intermediates in protein folding. Fragments of staphylococcal nuclease have been produced which also have properties similar to those of the molten globule, i.e. a somewhat compact structure with some secondary structure but without a defined tertiary structure. Pulsed hydrogen-deuterium exchange during refolding has been used to probe the protection of backbone amide hydrogens from solvent exchange during refolding of a number of proteins and most recently the staphylococcal nuclease Pro 117 yields Gly variant. The extent of exchange for 39 residues is determined by two-dimensional proton NMR after refolding for 5 ms to 10s. Three kinetic phases are inferred. Modest protection of amides in the early refolding intermediate composed to two beta-sheets formed by local sequence interactions is observed after a 5 ms refolding period. Native levels of protection throughout the molecule accrue more slowly in tow kinetic phases (k approximately 2s-1, k less than 0.01s-1). Protection factors were determined by varying the high pH labeling pulse after refolding for 100 ms. Little or no native or unfolded protein is present; instead, most molecules are in one or more partially folded states. The intermediate state has modest, yet significant, protection for residues in the beta-sheets (protection factors 10-60), and almost no protection in the alpha- helices (protection factors less than 10). The pattern of labeling is consistent with a role for beta turns and beta-hairpins in the formation of the early intermediate. Recently a chemical cleavage method has been developed where an EDT-Fe based reagent (EPD-Fe) can be attached to a protein via a cysteine side chain. The addition of ascorbate generates hydroxyl radicals at the iron center which diffuse and cleave the polypeptide backbone in a region close to the cysteine attachment site at residues accessible to solvent. The observed cleavage sites can be mapped by amino acid sequencing. The cleavage is dependent on protein conformation. We propose characterize the molten globule state of apomyoglobin and staphylococcal nuclease fragment structures using this newly developed chemical cleavage technique. We will prepare a number of cysteine variants of these proteins and characterize the cleavage patterns observed in the native and molten globule states.
虽然许多小的蛋白质可以在体外自发地折叠,但它们却可以在体外自动折叠

项目成果

期刊论文数量(5)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Directed discovery of bivalent peptide ligands to an SH3 domain.
直接发现 SH3 结构域的二价肽配体。
  • DOI:
    10.1110/ps.03470504
  • 发表时间:
    2004
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Ferguson,MoniqueR;Fan,Xiuzhen;Mukherjee,Munia;Luo,Jinquan;Khan,Raza;Ferreon,JosephineC;Hilser,VincentJ;Shope,RobertE;Fox,RobertO
  • 通讯作者:
    Fox,RobertO
The crystal structure of Escherichia coli heat shock protein YedU reveals three potential catalytic active sites.
大肠杆菌热休克蛋白 YedU 的晶体结构揭示了三个潜在的催化活性位点。
Stability studies of amino acid substitutions at tyrosine 27 of the staphylococcal nuclease beta-barrel.
葡萄球菌核酸酶 β-桶酪氨酸 27 处氨基酸取代的稳定性研究。
  • DOI:
    10.1021/bi970876r
  • 发表时间:
    1997
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Bhat,MG;Ganley,LM;Ledman,DW;Goodman,MA;Fox,RO
  • 通讯作者:
    Fox,RO
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Robert O. Fox其他文献

Letter to the Editor: Backbone and side chain resonance assignments of domain III of the tick-borne Langat flavivirus envelope protein
  • DOI:
    10.1023/b:jnmr.0000034345.94232.16
  • 发表时间:
    2004-08-01
  • 期刊:
  • 影响因子:
    1.900
  • 作者:
    Munia Mukherjee;Kaushik Dutta;Steven M. Pascal;Robert O. Fox
  • 通讯作者:
    Robert O. Fox
Mapping staphylococcal nuclease conformation using an EDTA-Fe derivative attached to genetically engineered cysteine residues.
使用连接到基因工程半胱氨酸残基的 EDTA-Fe 衍生物绘制葡萄球菌核酸酶构象。
  • DOI:
  • 发表时间:
    1994
  • 期刊:
  • 影响因子:
    2.9
  • 作者:
    M. Ermácora;D. W. Ledman;H. Hellinga;Gene W. Hsu;Robert O. Fox
  • 通讯作者:
    Robert O. Fox
Frederic Richards (1925–2009)
弗雷德里克·理查兹(1925 年至 2009 年)
  • DOI:
    10.1038/457976a
  • 发表时间:
    2009-02-18
  • 期刊:
  • 影响因子:
    48.500
  • 作者:
    Robert O. Fox
  • 通讯作者:
    Robert O. Fox

Robert O. Fox的其他文献

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{{ truncateString('Robert O. Fox', 18)}}的其他基金

Anti-tick-borne Encephalitis Virus Miniprotein Agents
抗蜱传脑炎病毒微蛋白制剂
  • 批准号:
    7760475
  • 财政年份:
    2009
  • 资助金额:
    $ 24.28万
  • 项目类别:
Anti-tick-borne Encephalitis Virus Miniprotein Agents
抗蜱传脑炎病毒微蛋白制剂
  • 批准号:
    6823388
  • 财政年份:
    2004
  • 资助金额:
    $ 24.28万
  • 项目类别:
Anti-tick-borne Encephalitis Virus Miniprotein Agents
抗蜱传脑炎病毒微蛋白制剂
  • 批准号:
    6932275
  • 财政年份:
    2004
  • 资助金额:
    $ 24.28万
  • 项目类别:
Anti-tick-borne Encephalitis Virus Miniprotein Agents
抗蜱传脑炎病毒微蛋白制剂
  • 批准号:
    7176235
  • 财政年份:
    2004
  • 资助金额:
    $ 24.28万
  • 项目类别:
Anti-tick-borne Encephalitis Virus Miniprotein Agents
抗蜱传脑炎病毒微蛋白制剂
  • 批准号:
    7015632
  • 财政年份:
    2004
  • 资助金额:
    $ 24.28万
  • 项目类别:
Anti-tick-borne Encephalitis Virus Miniprotein Agents
抗蜱传脑炎病毒微蛋白制剂
  • 批准号:
    7348316
  • 财政年份:
    2004
  • 资助金额:
    $ 24.28万
  • 项目类别:
Anti-tick-borne Encephalitis Virus Miniprotein Agents
抗蜱传脑炎病毒微蛋白制剂
  • 批准号:
    6677462
  • 财政年份:
    2003
  • 资助金额:
    $ 24.28万
  • 项目类别:
STRUCTURAL ANALYSIS OF PROTEIN FOLDING INTERMEDIATES
蛋白质折叠中间体的结构分析
  • 批准号:
    2022887
  • 财政年份:
    1997
  • 资助金额:
    $ 24.28万
  • 项目类别:
STRUCTURAL BIOLOGY OF THE GLYCINE RECEPTOR
甘氨酸受体的结构生物学
  • 批准号:
    6019282
  • 财政年份:
    1997
  • 资助金额:
    $ 24.28万
  • 项目类别:
STRUCTURAL ANALYSIS OF PROTEIN FOLDING INTERMEDIATES
蛋白质折叠中间体的结构分析
  • 批准号:
    2634753
  • 财政年份:
    1997
  • 资助金额:
    $ 24.28万
  • 项目类别:

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