Membrane dynamics are slowed for Alexa594-labeled membrane proteins due to substrate interactions
Membrane dynamics are slowed for Alexa594-labeled membrane proteins due to substrate interactions
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由于底物相互作用，Alexa594 标记的膜蛋白的膜动力学减慢
DOI：
10.1016/j.bbadva.2021.100026
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Knowles, Michelle K.
中科院分区：
文献类型：
--
作者：
Weisgerber, Alan W.;Knowles, Michelle K.
将荧光染料添加到蛋白质、脂质和其他生物分子中可以影响一系列过程，如迁移性、分子相互作用、定位，并最终影响功能。如果标签与底物或系统中的其他分子非特异性相互作用，则蛋白质的动力学会受到显著影响。为了测试染料-底物相互作用如何影响蛋白质扩散，设计了光漂白后荧光恢复（FRAP）测量，以明确确定染料对细胞表面表达的跨膜蛋白Syntaxin1a扩散的作用。Syntaxin1a在细胞外侧被EGFP标记，并附着一个带有或不带有染料标记的EGFP纳米体。在Syx1a-EGFP上进行了FRAP，在染料标记的纳米体存在下，细胞生长底物的选择影响了移动性。这项工作为使用Alexa594（一种用于红色荧光测量的常用探针）在FRAP和单分子跟踪测量中选择纤维连接蛋白（Fn）而不是聚l -赖氨酸（PLL）提供了证据。当细胞在PLL上培养时，alexa594标记的纳米体而不是未标记的纳米体显著降低了Syx1a-EGFP的流动性。然而，当使用Fn时，移动性恢复。单分子跟踪测量的迁移率与FRAP测量结果一致，Fn涂层表面比PLL更具迁移性。
The addition of fluorescent dyes to proteins, lipids and other biological molecules can affect a range of processes such as mobility, molecular interactions, localization, and, ultimately, function. The dynamics of a protein can be dramatically affected if the label interacts non-specifically with the substrate or with other molecules in the system. To test how dye-substrate interactions affect protein diffusion, fluorescence recovery after photobleaching (FRAP) measurements were designed to explicitly determine the role of the dye on the diffusion of a transmembrane protein, Syntaxin1a, expressed on the cell surface. Syntaxin1a, was tagged with EGFP on the extracellular side and an EGFP nanobody with or without a dye label was attached. FRAP was performed on Syx1a-EGFP and the choice of cell growth substrate affected mobility in the presence of a dye labeled nanobody. This work provides evidence for choosing fibronectin (Fn) over poly-L-lysine (PLL) in FRAP and single molecule tracking measurements when using Alexa594, a common probe for red fluorescent measurements. Alexa594-labeled nanobody but not unlabeled nanobody, dramatically reduced the mobility of Syx1a-EGFP when cells were cultured on PLL. However, when Fn was used, the mobility returned. Mobility measured by single molecule tracking measurements align with the FRAP measurements with Fn coated surfaces being more mobile than PLL.