title: Method for Developing Optical Sensors Using a Synthetic Dye-Fluorescent Protein FRET Pair and Computational Modeling and Assessment.
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.
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Mitchell
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: William H
authors.initials: WH
authors.lastname: Zhang
authors.affiliation: Institute of Cellular Neurosciences, University of Bonn Medical School, Bonn, Germany.
authors.forename: Michel K
authors.initials: MK
authors.lastname: Herde
authors.affiliation: Institute of Cellular Neurosciences, University of Bonn Medical School, Bonn, Germany.|German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany.|Institute of Neurology, University College London, London, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Henneberger
authors.affiliation: Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria.
authors.forename: Harald
authors.initials: H
authors.lastname: Janovjak
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Megan L
authors.initials: ML
authors.lastname: O'Mara
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia. colin.jackson@anu.edu.au.
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Jackson
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D003198:Computer Simulation; D003545:Cysteine / Q000235:genetics; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D003545:Cysteine
keywords: Computational modeling; Förster resonance energy transfer; Optical sensor; Synthetic dye