de Picciotto S1Dickson PM2Traxlmayr MW3Marques BS4Socher E5Zhao S1Cheung S5Kiefer JD6Wand AJ4Griffith LG7Imperiali B8Wittrup KD9. 2016. J Mol Biol. 428(20):4228-4241. doi: 10.1016/j.jmb.2016.07.004. Epub 2016 Jul 21.
1Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
2Department of Chemistry, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
3Koch Institute for Integrative Cancer Research, 500 Main Street, Cambridge, MA 02139, USA.
4Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
5Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 01239, USA.
6Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, Zurich, 8093, Switzerland.
7Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
8Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 01239, USA; Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
9Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, 500 Main Street, Cambridge, MA 02139, USA; Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address: wittrup@MIT.EDU.
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:

Sso7d scaffold; directed evolution; protein engineering; sensors; solvatochromism     

PMID:
 
27448945
 
PMCID:
 
PMC5048519
 [Available on 2017-10-09]
 
DOI:
 
10.1016/j.jmb.2016.07.004
[PubMed - in process]

*The Chicken anti-CMYC used in this publication is a product of Gallus Immunotech Inc.