Method for developing optical sensors using a synthetic dye-fluorescent protein FRET pair and computational modeling and assessment

Joshua A. Mitchell, William H. Zhang, Michel K. Herde, Christian Henneberger, Harald Janovjak, Megan L. O’Mara, Colin J. Jackson

Research output: Chapter in Book/Report/Conference proceedingChapter (Book)Otherpeer-review

Abstract

Biosensors that exploit Förster resonance energy transfer (FRET) can be used to visualize biological and physiological processes and are capable of providing detailed information in both spatial and temporal dimensions. In a FRET-based biosensor, substrate binding is associated with a change in the relative positions of two fluorophores, leading to a change in FRET efficiency that may be observed in the fluorescence spectrum. As a result, their design requires a ligand-binding protein that exhibits a conformational change upon binding. However, not all ligand-binding proteins produce responsive sensors upon conjugation to fluorescent proteins or dyes, and identifying the optimum locations for the fluorophores often involves labor-intensive iterative design or high-throughput screening. Combining the genetic fusion of a fluorescent protein to the ligand-binding protein with site-specific covalent attachment of a fluorescent dye can allow fine control over the positions of the two fluorophores, allowing the construction of very sensitive sensors. This relies upon the accurate prediction of the locations of the two fluorophores in bound and unbound states. In this chapter, we describe a method for computational identification of dye-attachment sites that allows the use of cysteine modification to attach synthetic dyes that can be paired with a fluorescent protein for the purposes of creating FRET sensors.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Pages89-99
Number of pages11
Volume1596
DOIs
Publication statusPublished - 2017
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1596
ISSN (Print)1064-3745

Keywords

  • Computational modeling
  • Förster resonance energy transfer
  • Optical sensor
  • Synthetic dye

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