Dynamics of Protein Assemblies by Analytical Ultracentrifugation

分析超速离心的蛋白质组装动力学

基本信息

项目摘要

One major thrust of our work has been the continued improvement of nonideal sedimentation velocity (nonideal SV) as a technique for measuring macromolecular size-distributions in highly concentrated solutions. The goal is to study proteins at concentrations closer to the intracellular environment, where weak interactions can govern a wide spectrum of behavior, including dynamic multi-protein complex formation and liquid-liquid phase transition. Our recent breakthrough in the analysis of polydisperse concentrated protein solutions came from the introduction of a mean-field approach to account for hydrodynamic interactions in the sedimentation particle mixtures. It is based on nonideality coefficients that arise from first-order approximations of concentration-dependent behavior, which, unfortunately, are limited to solutions with macromolecular volume occupancy below 5%. To remove this limitation, we have implemented higher-order approximations rooted in statistical fluid mechanics. In the past year, we have collected experimental data of sedimentation boundaries of bovine serum albumin at up to 150 mg/mL, and of undiluted serum. First results validate the sedimentation model. An experimental problem when working at high concentration is presented by optical aberrations in the strong refractive index gradients of the sedimentation boundary. This lensing effect can be minimized by short optical pathlengths, and therefore we have further improved the design of our 3D printed sample holders. We believe we have reached limits imposed by the need to seal the sample against high vacuum, achieve stability in high gravity, and the ability to create access ports for sample filling. We turned to a different approach to allow studying still higher protein concentrations: we have devised a technique for experimentally measuring the magnitude of optical aberrations, and developed a mathematical model of the optical distortions that we embedded it into our sedimentation data analysis. We have begun testing the performance of these approaches. A second major effort in our work on SV was directed at making sedimentation analysis more information-rich for the study of multi-protein interactions. We have previously pursued this goal by multi-signal approaches and creating temporal signals by photoswitching. However, another opportunity resides in the stratification of solution during the sedimentation process: Sedimenting systems of dynamically interacting proteins with complex life-times shorter than 1,000 sec exhibit richly patterned sedimentation profiles, with sedimentation boundaries that depend in complex ways on protein concentrations, equilibrium constants, and sedimentation coefficients associated with the different assembly states. We have previously developed effective particle theory that provides a physical explanation for sedimentation boundary patterns. In the current year we have integrated this theory into a new approach for interpreting SV data from sedimenting interacting systems. It allows taking advantage of the entire sedimentation pattern, rather than only the average sedimentation velocity, and thereby enhances the information content of SV experiments. We have implemented this into our data analysis software with a user interface that allows creation of customized binding models. Finally, we have continued to develop a fluorescence detection system for analytical ultracentrifugation, in collaboration with John Kakareka and Thomas Pohida (CIT). Both improvements in the optical setup as well as signal processing of index cells were achieved. To disseminate knowledge of analytical ultracentrifugation we have made this technique a major focus in our FEBS Practical Course that was held in January 2020 in Grenoble, France.
我们工作的一个主要推动力是继续改进非理想沉降速度(非理想SV)作为一种测量高浓度溶液中大分子尺寸分布的技术。 目标是研究浓度更接近细胞内环境的蛋白质,在细胞内环境中,弱相互作用可以控制广泛的行为,包括动态多蛋白质复合物的形成和液-液相转变。 我们最近在多分散浓缩蛋白质溶液的分析中的突破来自于引入平均场方法来解释沉降颗粒混合物中的流体动力学相互作用。 它是基于非理想性系数,从一阶近似的浓度依赖性行为,不幸的是,这是有限的解决方案与大分子体积占有率低于5%。 为了消除这种限制,我们已经实现了高阶近似植根于统计流体力学。在过去的一年中,我们收集了高达150 mg/mL的牛血清白蛋白和未稀释血清的沉降边界的实验数据。初步结果验证了沉降模型的正确性。 在高浓度下工作时的一个实验问题是在沉积边界的强折射率梯度中的光学像差。 这种透镜效应可以通过较短的光程长度来最小化,因此我们进一步改进了3D打印样品架的设计。我们相信,我们已经达到了对高真空密封样品、在高重力下实现稳定性以及创建样品填充入口的能力的要求所施加的极限。 我们转向了一种不同的方法来研究更高的蛋白质浓度:我们设计了一种实验测量光学畸变大小的技术,并开发了一种光学畸变的数学模型,我们将其嵌入到我们的沉降数据分析中。我们已经开始测试这些方法的性能。 我们在SV研究中的第二个主要努力是使沉降分析在多蛋白质相互作用的研究中信息更加丰富。 我们以前追求这一目标的多信号的方法,并通过光开关创建时间信号。 然而,另一个机会在于沉淀过程中溶液的分层:具有复杂生命期短于1,000秒的动态相互作用蛋白质的沉淀系统表现出丰富的模式化沉淀曲线,沉淀边界以复杂的方式依赖于与不同组装状态相关的蛋白质浓度,平衡常数和沉淀系数。我们以前开发的有效粒子理论,提供了一个物理解释的沉积边界模式。在本年度,我们已经整合了这一理论到一个新的方法来解释SV数据从沉积相互作用系统。 它允许利用整个沉积模式,而不仅仅是平均沉积速度,从而提高SV实验的信息内容。 我们已经在数据分析软件中实现了这一点,并提供了一个用户界面,允许创建定制的绑定模型。 最后,我们与John Kakareka和托马斯Pohida(CIT)合作,继续开发用于分析超离心的荧光检测系统。 实现了光学设置以及折射率单元的信号处理的改进。 为了传播分析超离心的知识,我们将这项技术作为2020年1月在法国格勒诺布尔举行的FEBS实践课程的重点。

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:
    $ 31.9万
  • 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:
    $ 31.9万
  • 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:
    $ 31.9万
  • 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:
    $ 31.9万
  • 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:
    $ 31.9万
  • 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:
    $ 31.9万
  • 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:
    $ 31.9万
  • 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:
    9361484
  • 财政年份:
  • 资助金额:
    $ 31.9万
  • 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:
    $ 31.9万
  • 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:
    $ 31.9万
  • 项目类别:

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