title: A peptide-based, ratiometric biosensor construct for direct fluorescence detection of a protein analyte.
abstract: We present the design, synthesis, and functional evaluation of peptide-based fluorescent constructs for wavelength-ratiometric biosensing of a protein analyte. The concept was shown using the high-affinity model interaction between the 18 amino acid peptide pTMVP and a recombinant antibody fragment, Fab57P. pTMVP was functionalized in two different positions with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive fluorophore with a two-band emission. The equilibrium dissociation constant of the interaction between pTMVP and Fab57P was largely preserved upon labeling. The biosensor ability of the labeled peptide constructs was evaluated in terms of the relative intensity change of the emission bands from the normal (N*) and tautomer (T*) excited-state species of the fluorophore ( I(N*)/I(T*)) upon binding of Fab57P. When the peptide was labeled in the C terminus, the I(N*)/I(T*) ratio changed by 40% upon analyte binding, while labeling close to the residues most important for binding resulted in a construct that completely lacked ratiometric biosensor ability. Integrated biosensor elements for reagentless detection, where peptides and ratiometric fluorophores are combined to ensure robustness in both recognition and signaling, are expected to become an important contribution to the design of future protein quantification assays in immobilized formats.
authors.affiliation: Division of Molecular Physics, Department of Physics, Chemistry and Biology, Linkoping University, 58183 Linkoping, Sweden. karen@ifm.liu.se
authors.forename: Karin
authors.initials: K
authors.lastname: Enander
authors.forename: Laurence
authors.initials: L
authors.lastname: Choulier
authors.forename: A Linnéa
authors.initials: AL
authors.lastname: Olsson
authors.forename: Dmytro A
authors.initials: DA
authors.lastname: Yushchenko
authors.forename: Daniel
authors.initials: D
authors.lastname: Kanmert
authors.forename: Andrey S
authors.initials: AS
authors.lastname: Klymchenko
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Demchenko
authors.forename: Yves
authors.initials: Y
authors.lastname: Mély
authors.forename: Danièle
authors.initials: D
authors.lastname: Altschuh
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D010455:Peptides; D011506:Proteins