Method for Developing Optical Sensors Using a Synthetic Dye-Fluorescent Protein FRET Pair and Computational Modeling and Assessment

Date

2017

Authors

Mitchell, Joshua
Zhang, William
Herde, Michel K.
Henneberger, Christian
Janovjak, Harald
O'Mara, Megan
Jackson, Colin

Journal Title

Journal ISSN

Volume Title

Publisher

Humana Press

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.

Description

Keywords

Citation

Source

Type

Book chapter

Book Title

Synthetic Protein Switches : Methods and Protocols

Entity type

Access Statement

License Rights

DOI

10.1007/978-1-4939-6940-1_6

Restricted until

2099-12-31