PROTEIN FOLDING DYNAMICS BY MASS SPECTROMETRY

通过质谱分析蛋白质折叠动力学

基本信息

  • 批准号:
    6387202
  • 负责人:
  • 金额:
    $ 17.49万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2000
  • 资助国家:
    美国
  • 起止时间:
    2000-07-01 至 2005-06-30
  • 项目状态:
    已结题

项目摘要

Elucidation of the mechanisms by which proteins fold to attain their native structure remains one of the most challenging problems in biochemistry. Recently, many diseases have been linked to possible protein misfolding followed by a loss/alteration of its function and/or aggregation (amyloidosis). There is also a very close relationship between the folding/conformational stability of proteins and their ligand- binding properties. We are seeking to further our understanding of the dynamics of the folding process, as well as conformational stability of proteins and protein-ligand complexes. To that end, we will use electrospray (ESI) mass spectrometry (MS) to study folding processes in vitro. We will employ hydrogen/deuterium (H/D) exchange in solution to probe the conformational stability and folding/unfolding dynamics of proteins under various conditions. Transiently populated intermediate states will be detected in these experiments and characterized by different degrees of backbone amide protection. Because of the high data acquisition rate, mass spectrometry offers a facile way to resolve these intermediates on a time scale from tens of msec to hours. The global information on the protein conformational stability and folding dynamics will be complemented by local (residue-specific) information. Several approaches will be used to achieve that goal. In the first approach, collisionally activated dissociation (CAD) of protein ions will be used to measure the deuterium content locally as a function of exchange time. Particular care will be taken to ensure that ion-molecular processes in the gas phase (e.g., hydrogen scrambling and/or H/D exchange with residual solvent molecules) do not influence the measurements. This approach will be particularly useful for studying the folding events on a time scale of tens of seconds to hours, as it allows real time monitoring to be carried out. In another approach, the HID exchange will be quenched at a certain time (by lowering the solution pH and temperature) and extent of the H/D exchange will be analyzed by using ESI/CAD or peptic digest followed by ESI MS analysis. This will allow us to extend our studies to a sub-second time scale. We will also use chemical cross-linking to elucidate the topology of ligand binding sites in proteins, as well as inter-domain interactions in multi-domain proteins. These methods will be applied to study folding dynamics and conformational stability of several proteins. Molecular mechanisms of ligand binding to Cellular Retinoic Acid Binding Protein I (CRABP I) will be studied by mapping the energy landscape for both apo- and holo-forms of the protein. We will also study mechanisms of ferric ion binding/release by iron transporting proteins from the transferrin family. The proposed studies will significantly further our understanding of the protein folding processes and the mechanisms by which transport proteins function in vivo.
阐明蛋白质折叠以获得其天然结构的机制仍然是生物化学中最具挑战性的问题之一。 最近,许多疾病已经与可能的蛋白质错误折叠,随后其功能和/或聚集的丧失/改变(淀粉样变性)有关。蛋白质的折叠/构象稳定性与其配体结合性质之间也存在非常密切的关系。我们正在寻求进一步了解的折叠过程的动力学,以及蛋白质和蛋白质-配体复合物的构象稳定性。 为此,我们将使用电喷雾(ESI)质谱(MS)研究体外折叠过程。 我们将采用氢/氘(H/D)交换在溶液中探测蛋白质的构象稳定性和折叠/解折叠动力学在各种条件下。在这些实验中将检测到瞬时填充的中间状态,并通过不同程度的骨架酰胺保护来表征。 由于高数据采集速率,质谱法提供了一种简便的方法来解决这些中间体的时间尺度从几十毫秒到几个小时。 关于蛋白质构象稳定性和折叠动力学的全局信息将由局部(残基特异性)信息补充。 为实现这一目标,将采取若干办法。 在第一种方法中,蛋白质离子的碰撞激活解离(CAD)将被用来测量局部的氘含量作为交换时间的函数。将特别注意确保气相中的离子-分子过程(例如,氢扰乱和/或与残留溶剂分子的H/D交换)不影响测量。 这种方法对于研究数十秒至数小时的时间尺度上的折叠事件特别有用,因为它允许进行真实的时间监测。 在另一种方法中,HID交换将在某个时间被淬灭(通过降低溶液pH和温度),并且H/D交换的程度将通过使用ESI/CAD或消化酶消化,随后进行ESI MS分析来分析。 这将使我们能够将我们的研究扩展到亚秒的时间尺度。 我们还将使用化学交联来阐明蛋白质中配体结合位点的拓扑结构,以及多结构域蛋白质中的结构域间相互作用。这些方法将被应用于研究几种蛋白质的折叠动力学和构象稳定性。 配体结合细胞视黄酸结合蛋白I(CRABP I)的分子机制将通过绘制蛋白质的脱辅基和全形式的能量景观来研究。 我们还将研究转铁蛋白家族铁转运蛋白结合/释放三价铁离子的机制。拟议的研究将显着进一步我们的蛋白质折叠过程和转运蛋白在体内发挥作用的机制的理解。

项目成果

期刊论文数量(0)
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IGOR A KALTASHOV其他文献

IGOR A KALTASHOV的其他文献

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{{ truncateString('IGOR A KALTASHOV', 18)}}的其他基金

Cross-path reactive chromatography/mass spectrometry as a versatile platform for characterization of primary and higher order structure of complex heterogeneous proteins
交叉路径反应色谱/质谱作为多功能平台,用于表征复杂异质蛋白质的一级和高级结构
  • 批准号:
    10350609
  • 财政年份:
    2019
  • 资助金额:
    $ 17.49万
  • 项目类别:
An integrated mass spectrometry approach to study heparin structure-bioactivity
研究肝素结构-生物活性的综合质谱方法
  • 批准号:
    9252476
  • 财政年份:
    2016
  • 资助金额:
    $ 17.49万
  • 项目类别:
An integrated mass spectrometry approach to study heparin structure-bioactivity
研究肝素结构-生物活性的综合质谱方法
  • 批准号:
    10531619
  • 财政年份:
    2016
  • 资助金额:
    $ 17.49万
  • 项目类别:
An integrated mass spectrometry approach to study heparin structure-bioactivity
研究肝素结构-生物活性的综合质谱方法
  • 批准号:
    10322743
  • 财政年份:
    2016
  • 资助金额:
    $ 17.49万
  • 项目类别:
Investigation of protein dynamics by mass spectrometry
通过质谱研究蛋白质动力学
  • 批准号:
    7935574
  • 财政年份:
    2009
  • 资助金额:
    $ 17.49万
  • 项目类别:
ACQUISITION ELECTROSPRAY TOF MASS SPECTROMETER: PROTEIN STUDIES
采集电喷雾 TOF 质谱仪:蛋白质研究
  • 批准号:
    6973452
  • 财政年份:
    2004
  • 资助金额:
    $ 17.49万
  • 项目类别:
Acquisition of an electrospray TOF mass spectrometer
购置电喷雾 TOF 质谱仪
  • 批准号:
    6732556
  • 财政年份:
    2004
  • 资助金额:
    $ 17.49万
  • 项目类别:
Investigation of protein dynamics by mass spectrometry
通过质谱研究蛋白质动力学
  • 批准号:
    7254052
  • 财政年份:
    2000
  • 资助金额:
    $ 17.49万
  • 项目类别:
Investigations of interactions in dynamic protein complexes by mass spectrometry
通过质谱研究动态蛋白质复合物中的相互作用
  • 批准号:
    8310015
  • 财政年份:
    2000
  • 资助金额:
    $ 17.49万
  • 项目类别:
PROTEIN FOLDING DYNAMICS BY MASS SPECTROMETRY
通过质谱分析蛋白质折叠动力学
  • 批准号:
    6520291
  • 财政年份:
    2000
  • 资助金额:
    $ 17.49万
  • 项目类别:

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Characterization of the Transition State Ensemble for Protein Folding Using Molecular Dynamics Calculations of Molecular Volumes
使用分子体积的分子动力学计算表征蛋白质折叠的过渡态系综
  • 批准号:
    410809-2011
  • 财政年份:
    2011
  • 资助金额:
    $ 17.49万
  • 项目类别:
    Alexander Graham Bell Canada Graduate Scholarships - Master's
MOLECULAR DYNAMICS SIMULATIONS OF PROTEIN FOLDING
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  • 批准号:
    8363653
  • 财政年份:
    2011
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    $ 17.49万
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Development of new molecular dynamics simulation methods and application to protein folding problem
新分子动力学模拟方法的发展及其在蛋白质折叠问题中的应用
  • 批准号:
    23740325
  • 财政年份:
    2011
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    $ 17.49万
  • 项目类别:
    Grant-in-Aid for Young Scientists (B)
LONG TIMESCALE MOLECULAR DYNAMICS SIMULATION OF PROTEIN FOLDING
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  • 批准号:
    8364335
  • 财政年份:
    2011
  • 资助金额:
    $ 17.49万
  • 项目类别:
MOLECULAR DYNAMICS SIMULATIONS OF PROTEIN FOLDING
蛋白质折叠的分子动力学模拟
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  • 财政年份:
    2010
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    $ 17.49万
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MOLECULAR DYNAMICS SIMULATIONS OF PROTEIN FOLDING
蛋白质折叠的分子动力学模拟
  • 批准号:
    7955611
  • 财政年份:
    2009
  • 资助金额:
    $ 17.49万
  • 项目类别:
SIMULATION OF PROTEIN FOLDING INTERMEDIATES USING MOLECULAR DYNAMICS
使用分子动力学模拟蛋白质折叠中间体
  • 批准号:
    6221121
  • 财政年份:
    1999
  • 资助金额:
    $ 17.49万
  • 项目类别:
SIMULATION OF PROTEIN FOLDING INTERMEDIATES USING MOLECULAR DYNAMICS
使用分子动力学模拟蛋白质折叠中间体
  • 批准号:
    6282525
  • 财政年份:
    1998
  • 资助金额:
    $ 17.49万
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SIMULATION OF PROTEIN FOLDING INTERMEDIATES USING MOLECULAR DYNAMICS
使用分子动力学模拟蛋白质折叠中间体
  • 批准号:
    6122490
  • 财政年份:
    1998
  • 资助金额:
    $ 17.49万
  • 项目类别:
SIMULATION OF PROTEIN FOLDING INTERMEDIATES USING MOLECULAR DYNAMICS
使用分子动力学模拟蛋白质折叠中间体
  • 批准号:
    6295180
  • 财政年份:
    1998
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    $ 17.49万
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