de Picciotto Seymour, Dickson Paige M, Traxlmayr Michael W, Marques Bryan S, Socher Elke, Zhao Sixing, Cheung Stephanie, Kiefer Jonathan D, Wand A Joshua, Griffith Linda G, Imperiali Barbara, Wittrup K Dane
Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Department of Chemistry, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
J Mol Biol. 2016 Oct 9;428(20):4228-4241. doi: 10.1016/j.jmb.2016.07.004. Epub 2016 Jul 21.
Quantifying protein location and concentration is critical for understanding function in situ. Scaffold conjugated to environment-sensitive fluorophore (SuCESsFul) biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can, in principle, detect analytes of interest with high temporal and spatial resolution. However, their adoption has been limited due to the extensive empirical screening required for their development. We sought to establish design principles for this class of biosensor by characterizing over 400 biosensors based on various protein analytes, binding proteins, and fluorophores. We found that the brightest readouts are attained when a specific binding pocket for the fluorophore is present on the analyte. Also, interaction of the fluorophore with the binding protein it is conjugated to can raise background fluorescence, considerably limiting sensor dynamic range. Exploiting these two concepts, we designed biosensors that attain a 100-fold increase in fluorescence upon binding to analyte, an order of magnitude improvement over the previously best-reported SuCESsFul biosensor. These design principles should facilitate the development of improved SuCESsFul biosensors.
量化蛋白质的位置和浓度对于理解其原位功能至关重要。与环境敏感荧光团结合的支架（SuCESsFul）生物传感器，即将报告荧光团与结合支架相连，原则上能够以高时空分辨率检测感兴趣的分析物。然而，由于其开发需要广泛的经验筛选，这类传感器的应用受到了限制。我们试图通过对基于各种蛋白质分析物、结合蛋白和荧光团的400多种生物传感器进行表征，来确立这类生物传感器的设计原则。我们发现，当分析物上存在荧光团的特定结合口袋时，能够获得最亮的读数。此外，荧光团与其相连的结合蛋白之间的相互作用会增加背景荧光，从而极大地限制了传感器的动态范围。利用这两个概念，我们设计出了与分析物结合时荧光增强100倍的生物传感器，比之前报道的最佳SuCESsFul生物传感器有了一个数量级的提升。这些设计原则应有助于改进型SuCESsFul生物传感器的开发。
