Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield
Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield
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DOI：
10.1016/j.bpj.2017.02.020
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发表时间：
2017-04-11
影响因子：
3.4
通讯作者：
Schuck, Peter
中科院分区：
文献类型：
--
作者：
Chaturvedi, Sumit K.;Zhao, Huaying;Schuck, Peter
结合荧光检测的沉降速度分析超离心已成为研究大分子相互作用体系的一种强有力的方法。它结合了皮摩尔灵敏度和高流体动力学分辨率，可以使用可切换的荧光团进行多组分鉴别，以确定具有从皮摩尔到微摩尔解离平衡常数的大分子配合物的化学计量、亲和力和形状。一种流行的数据解释方法是通过重量平均沉降系数sw的等温线来确定结合亲和力。沉积分析中一个流行的教条是，从输运法得到的加权平均沉积系数对应于所有物种的信号和种群加权平均值。我们表明，对于那些表现出具有复杂形成特性的显著信号变化的系统，这并不总是成立的，这些特性在实践中可能很容易遇到，例如，荧光量子产率的变化。在快速可逆系统的反应边界中的耦合输运可以对观测到的迁移做出重大贡献，这在标准的基于种群的平均值中是无法解释的。有效粒子理论为反应耦合迁移过程提供了一个简单的物理图景。在此基础上，我们开发了一个更通用的结合模型，该模型收敛于众所周知的具有恒定信号的sw形式，但可以同时考虑荧光量子产率变化时的流体动力学共输运。我们相信这将有助于研究具有荧光猝灭、增强或福斯特共振能量传递的相互作用系统。
Sedimentation velocity analytical ultracentrifugation with fluorescence detection has emerged as a powerful method for the study of interacting systems of macromolecules. It combines picomolar sensitivity with high hydrodynamic resolution, and can be carried out with photoswitchable fluorophores for multicomponent discrimination, to determine the stoichiometry, affinity, and shape of macromolecular complexes with dissociation equilibrium constants from picomolar to micromolar. A popular approach for data interpretation is the determination of the binding affinity by isotherms of weight-average sedimentation coefficients sw. A prevailing dogma in sedimentation analysis is that the weight-average sedimentation coefficient from the transport method corresponds to the signal-and population-weighted average of all species. We show that this does not always hold true for systems that exhibit significant signal changes with complex formation-properties that may be readily encountered in practice, e.g., from a change in fluorescence quantum yield. Coupled transport in the reaction boundary of rapidly reversible systems can make significant contributions to the observed migration in a way that cannot be accounted for in the standard population-based average. Effective particle theory provides a simple physical picture for the reaction-coupled migration process. On this basis, we develop a more general binding model that converges to the well-known form of sw with constant signals, but can account simultaneously for hydrodynamic cotransport in the presence of changes in fluorescence quantum yield. We believe this will be useful when studying interacting systems exhibiting fluorescence quenching, enhancement, or Forster resonance energy transfer with transport methods.