89
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_6, © Springer Science+Business Media LLC 2017
Chapter 6
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,
and Colin J. Jackson
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.
Key words Synthetic dye, Optical sensor, Computational modeling, Förster resonance energy transfer
1 Introduction
Optical sensors have allowed for the investigation of physiological
processes such as neurotransmission with both spatial and temporal resolution. FRET-based optical sensors are particularly useful,
as they are capable of giving quantitative recordings independent
of sensor concentration due to the ratiometric signal output of the
sensor and the concentration independence of FRET donor
lifetimes [1, 2]. Contemporary sensors typically use fluorescent
proteins (FPs). However, some have used synthetic fluorescent
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_6, © Springer Science+Business Media LLC 2017
Chapter 6
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,
and Colin J. Jackson
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.
Key words Synthetic dye, Optical sensor, Computational modeling, Förster resonance energy transfer
1 Introduction
Optical sensors have allowed for the investigation of physiological
processes such as neurotransmission with both spatial and temporal resolution. FRET-based optical sensors are particularly useful,
as they are capable of giving quantitative recordings independent
of sensor concentration due to the ratiometric signal output of the
sensor and the concentration independence of FRET donor
lifetimes [1, 2]. Contemporary sensors typically use fluorescent
proteins (FPs). However, some have used synthetic fluorescent