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


Author(s): Mitchell, Joshua A; Zhang, William H; Herde, Michel K; Henneberger, Christian; Janovjak, Harald; O'Mara, Megan L; Jackson, Colin J
Article/Chapter Title: Method for developing optical sensors using a synthetic dye fluorescent protein FRET pair and computational modeling and assessment
Title Series: Methods in Molecular Biology
Affiliation IST Austria
Abstract: Biosensors that exploit Forster 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.
Keywords: Computational modeling; Synthetic dye; optical sensor; Förster resonance energy transfer
Book Title: Synthetic Protein Switches
Volume: 1596
ISBN: 978-1-4939-6940-1
Publisher: Springer  
Date Published: 2017-05-15
Start Page: 89
End Page: 99
DOI: 10.1007/978-1-4939-6940-1_6
Open access: no