Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions
Name
nihms804841.pdf
Size
1.26 MB
Format
Adobe PDF
Checksum (MD5)
b87e6e5e19bcf29fb07485d32a781c04
Author(s)
Dickson, Paige M.
Marques, Bryan S.
Zhao, Sixing
Kiefer, Jonathan D.
Wand, A. Joshua
Griffith, Linda G.
De Picciotto, Seymour
Traxlmayr, Michael
Socher, Elke Ricarda
Cheung, Stephanie
Date Issued
July 2016
Journal
Journal of Molecular Biology
Publisher
Elsevier BV
Citation
De Picciotto, Seymour et al. “Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions.” Journal of Molecular Biology 428, 20 (October 2016): 4228–4241 © 2016 Elsevier Ltd
Version
Author's final manuscript
Abstract
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. Keywords: solvatochromism; Sso7d scaffold; sensors; protein engineering; directed evolution
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemical Engineering
Massachusetts Institute of Technology. Department of Chemistry
Koch Institute for Integrative Cancer Research at MIT
Terms of Use
Creative Commons Attribution-NonCommercial-NoDerivs License
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1016/J.JMB.2016.07.004
