Biophysical Characterization Of Macromolecules

大分子的生物物理表征

基本信息

项目摘要

The intent of this project is to develop new and to improve existing biophysical methodology for the characterization of biological macromolecules in solution, and to apply these methods collaboratively to the study of proteins and their interactions. Experimental techniques employed are analytical ultracentrifugation, static and dynamic light scattering, isothermal titration calorimetry, differential scanning calorimetry, circular dichroism spectroscopy, and surface plasmon resonance biosensing. For the study of reversible formation of multi-protein complexes in solution, we have previously developed a novel multi-signal analysis for sedimentation velocity analytical ultracentrifugation. It can permit the label-free detection of sedimentation coefficient distributions of currently up to three protein components, and thus measure the stoichiometry of multiple hydrodynamically separated protein complexes. We have proceeded from the development stage of this method to the collaborative application in several systems. This includes the study of the heterogeneous triple protein complexes discovered for adaptor protein complexes involved in signal transduction after T-cell activation, as well as a study on the ternary interactions of cell surface receptors with HIV envelope protein. We have made significant progress in the theoretical models to account for the influence of reaction kinetics on the sedimentation behavior of interacting systems. This has permitted the more reliable hydrodynamic characterization of proteins by sedimentation velocity analytical ultracentrifugation. This could be applied to collaborative studies of several viral proteins, including the malarial surface protein MSP-3, clathrin basked assembly, the study of the oligomeric state and detergent-binding of detergent-solubilized CB2 receptor, and the interaction of iron-regulatory proteins IRP1 and IRP2 with the iron regulatory elements. With the goal to study conformational heterogeneity and ligand-induced conformational changes of proteins, we have embarked on the adaptation of a difference sedimentation technique to modern Rayleigh interference and absorption optical detection systems. This has promise to increase the sensitivity over existing traditional approaches. We have identified limiting factors in the detection, and developed a protocol for a suitable optical alignment. In the area of optical biosensing, we have further explored the properties of our computational approach to extract information on the functional heterogeneity of surface binding sites from experimental data. This was applied to the characterization of antibody-antigen interactions, and the analysis of integrin receptor with various ligands. In continuation of our studies on small molecule binding to proteins, we have applied surface plasmon resonance (SPR) spectroscopy to characterize new inhibitors of HIV reverse transcriptase and human RNAse H. We have explored the study of protein/ligand stoichiometry by SPR spectroscopy, and the influence of small molecule ligand binding on the protein self-association by sedimentation velocity analytical ultracentrifugation.
这个项目的目的是开发新的和改进现有的生物物理方法来表征溶液中的生物大分子,并将这些方法共同应用于蛋白质及其相互作用的研究。所采用的实验技术有分析超速离心法、静态和动态光散射、等温滴定量热法、差示扫描量热法、圆二色谱和表面等离子体共振生物传感。 为了研究多蛋白质复合体在溶液中的可逆形成,我们以前发展了一种新的多信号分析方法,用于沉降速度分析的超速离心法。它可以实现对目前最多三个蛋白质组分的沉降系数分布的无标记检测,从而测量多个流体动力学分离的蛋白质复合体的化学计量比。我们已经从该方法的开发阶段进展到在多个系统中的协同应用。这包括对T细胞激活后参与信号转导的接头蛋白复合体的异质三蛋白复合体的研究,以及对细胞表面受体与HIV包膜蛋白三元相互作用的研究。 我们在考虑反应动力学对相互作用体系沉淀行为的影响的理论模型方面取得了重大进展。这使得通过沉降速度分析超速离心法对蛋白质进行更可靠的流体动力学表征成为可能。这可用于几种病毒蛋白的合作研究,包括疟疾表面蛋白MSP-3、笼蛋白洗涤组装、洗涤剂增溶的CB2受体的寡聚态和洗涤剂结合的研究,以及铁调节蛋白Irp1和IRP2与铁调节元件的相互作用。 为了研究蛋白质的构象异质性和配体诱导的构象变化,我们已经着手将一种差异沉淀技术应用于现代瑞利干涉和吸收光学检测系统。这有望提高对现有传统方法的敏感性。我们已经确定了检测中的限制因素,并开发了一种合适的光学对准方案。 在光学生物传感领域,我们进一步探索了我们的计算方法的特性,以从实验数据中提取关于表面结合位点功能异质性的信息。这被应用于抗体-抗原相互作用的表征,以及整合素受体与不同配体的分析。 在我们对小分子与蛋白质结合的研究中,我们应用表面等离子体共振(SPR)光谱表征了新型HIV逆转录酶和人核糖核酸酶H的抑制剂。我们探索了SPR光谱对蛋白质/配体化学计量比的研究,以及通过沉降速度分析超速离心法研究小分子配体结合对蛋白质自结合的影响。

项目成果

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PETER SCHUCK其他文献

PETER SCHUCK的其他文献

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{{ truncateString('PETER SCHUCK', 18)}}的其他基金

BIOPHYSICAL CHARACTERIZATION OF MACROMOLECULES
大分子的生物物理表征
  • 批准号:
    6290696
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Dynamics of Protein Assemblies by Analytical Ultracentrifugation
分析超速离心的蛋白质组装动力学
  • 批准号:
    8743775
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Multi-Method Approaches for the Study of Complex Protein Interactions
研究复杂蛋白质相互作用的多种方法
  • 批准号:
    8933882
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Dynamics of Protein Assemblies by Analytical Ultracentrifugation
分析超速离心的蛋白质组装动力学
  • 批准号:
    10262996
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Higher-Order Structure and Solution Interactions of Antibodies
抗体的高阶结构和溶液相互作用
  • 批准号:
    10263002
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Interactions of SARS-CoV-2 N-protein
SARS-CoV-2 N 蛋白的相互作用
  • 批准号:
    10263005
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Biophysical Characterization Of Macromolecules
大分子的生物物理表征
  • 批准号:
    7967861
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Multi-Method Approaches for the Study of Complex Protein Interactions
研究复杂蛋白质相互作用的多种方法
  • 批准号:
    7734387
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Development of Biosensor Technology for Protein Interactions
蛋白质相互作用生物传感器技术的发展
  • 批准号:
    7967910
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Dynamics of Protein Assemblies by Analytical Ultracentrifugation
分析超速离心的蛋白质组装动力学
  • 批准号:
    8340624
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:

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禽类植入式抗原抗体反应传感器的研制
  • 批准号:
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    15591668
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DEVELOPMENT OF THE INTRAOPERATIVE ESTIMATION OF THE PROXIMAL NERVE STUMP USING ANTIGEN-ANTIBODY REACTION ON THE ARTIFICIAL MEMBRANE
利用人工膜上抗原抗体反应进行近端神经残端术中估计的研究进展
  • 批准号:
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    --
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    --
  • 项目类别:
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