Accounting for photophysical processes and specific signal intensity changes in fluorescence-detected sedimentation velocity.-猫眼课题宝

DOI：

10.1021/ac502478a

发表时间：

2014-09-16

期刊：

ANALYTICAL CHEMISTRY

影响因子：

7.4

通讯作者：

中科院分区：

化学1区

文献类型：

作者：

Zhao, Huaying;Ma, Jia;Ingaramo, Maria;Andrade, Eric;MacDonald, Jeff;Ramsay, Glen;Piszczek, Grzegorz;Patterson, George H.;Schuck, Peter

来源链接：

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4165462/?report=classic

荧光检测沉降速度 (FDS-SV) 已成为研究高亲和力蛋白质相互作用的强大技术，其流体动力学分辨率超过基于扩散的技术，并且对于低皮摩尔浓度下的结合研究具有足够的灵敏度。为了对观测到的沉积边界进行详细的定量分析，需要将常规的沉积模型调整为FDS数据结构。一个关键的考虑因素是实验过程中大分子荧光强度的变化，这是由激发激光功率的缓慢漂移和/或光物理过程引起的。在目前的工作中，我们证明 FDS-SV 数据本质上具有时间依赖性大分子信号强度的参考，依赖于径向边界迁移和平台信号之间的几何联系。我们展示了如何利用这个新的时域来研究表现出光漂白和光活化的分子。这将 FDS-SV 的应用扩展到带有光开关荧光蛋白、有机染料或纳米颗粒标记的蛋白质，例如最近引入的亚衍射显微镜，并使 FDS-SV 研究它们的相互作用和尺寸分布成为可能。同时，我们发现传统荧光团在 FDS 的标准照明下经历最小的光漂白。这些发现支持应用高激光功率密度进行检测，我们证明这可以进一步提高信号质量。

Fluorescence detected sedimentation velocity (FDS-SV) has emerged as a powerful technique for the study of high-affinity protein interactions, with hydrodynamic resolution exceeding that of diffusion-based techniques, and with sufficient sensitivity for binding studies at low picomolar concentrations. For the detailed quantitative analysis of the observed sedimentation boundaries, it is necessary to adjust the conventional sedimentation models to the FDS data structure. A key consideration is the change in the macromolecular fluorescence intensity during the course of the experiment, caused by slow drifts of the excitation laser power, and/or by photophysical processes. In the present work, we demonstrate that FDS-SV data have inherently a reference for the time-dependent macromolecular signal intensity, resting on a geometric link between radial boundary migration and plateau signal. We show how this new time-domain can be exploited to study molecules exhibiting photobleaching and photoactivation. This expands the application of FDS-SV to proteins tagged with photoswitchable fluorescent proteins, organic dyes, or nanoparticles, such as those recently introduced for subdiffraction microscopy and enables FDS-SV studies of their interactions and size distributions. At the same time, we find that conventional fluorophores undergo minimal photobleaching under standard illumination in the FDS. These findings support the application of a high laser power density for the detection, which we demonstrate can further increase the signal quality.