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Carnegie Institution, Stanford, California 94305, USA
(RECEIVED April 27, 2005; FINAL REVISION June 17, 2005; ACCEPTED June 18, 2005)
A family of genetically-encoded metabolite sensors has been constructed using bacterial periplasmic binding proteins (PBPs) linearly fused to protein fluorophores. The ligand-induced conformational change in a PBP allosterically regulates the relative distance and orientation of a fluorescence resonance energy transfer (FRET)-compatible protein pair. Ligand binding is transduced into a macroscopic FRET observable, providing a reagent for in vitro and in vivo ligand-measurement and visualization. Sensors with a higher FRET signal change are required to expand the dynamic range and allow visualization of subtle analyte changes under high noise conditions. Various observations suggest that factors other than inter-fluorophore separation contribute to FRET transfer efficiency and the resulting ligand-dependent spectral changes. Empirical and rational protein engineering leads to enhanced allosteric linkage between ligand binding and chromophore rearrangement; modifications predicted to decrease chromophore rotational averaging enhance the signal change, emphasizing the importance of the rotational freedom parameter 
2 to FRET efficiency. Tighter allosteric linkage of the PBP and the fluorophores by linker truncation or by insertion of chromophores into the binding protein at rationally designed sites gave rise to sensors with improved signal change. High-response sensors were obtained with fluorescent proteins attached to the same binding PBP lobe, suggesting that indirect allosteric regulation during the hinge-bending motion is sufficient to give rise to a FRET response. The optimization of sensors for glucose and glutamate, ligands of great clinical interest, provides a general framework for the manipulation of ligand-dependent allosteric signal transduction mechanisms.
Keywords: conformational changes; structure/function studies; new methods; molecular mechanics/dynamics; glucose; glutamate; nanosensor; neurotransmitter; biosensor; protein engineering; FRET
Abbreviations: FLIPglu, fluorescent indicator protein for glucose • FLIPE, fluorescent indicator protein for glutamate • FRET, fluorescence resonance energy transfer • FP, fluorescent protein • ECFP, enhanced cyan fluorescent protein • EYFP, enhanced yellow fluorescent protein • BP, binding protein • PBP, periplasmic binding protein
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051508105.
Reprint requests to: Wolf B. Frommer, Carnegie Institution, 260 Panama Street, Stanford, CA 94305, USA; e-mail: wfrommer{at}stanford.edu; fax: (650) 325-6857.
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