title: Quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide linkers.
abstract: The fusion of different protein domains via peptide linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP-linker-EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistence length of 4.5 A or a Gaussian chain with a characteristic ratio of 2.3. The implications of our results for understanding the properties of FRET-based sensors and other fusion proteins with Gly/Ser linkers are discussed.
authors.affiliation: Laboratory of Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology, Post Office Box 513, 5600 MB Eindhoven, The Netherlands.
authors.forename: Toon H
authors.initials: TH
authors.lastname: Evers
authors.forename: Elisabeth M W M
authors.initials: EM
authors.lastname: van Dongen
authors.forename: Alex C
authors.initials: AC
authors.lastname: Faesen
authors.forename: E W
authors.initials: EW
authors.lastname: Meijer
authors.forename: Maarten
authors.initials: M
authors.lastname: Merkx
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D004247:DNA; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D010455:Peptides; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D004247:DNA