Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering

Abstract

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 k 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 Supplemental material: see www.proteinscience.org Biosensors are molecular machines linking the highly evolved ligand-binding properties of a biological macromolecule to the production of a physical observable (Nakamura and Karube 2003;Looger et al. 2005). Many classes of biomolecules have been employed as ''recognition elements'' of biosensors, although proteins have emerged as the gold standard due to their superior properties, including specificity, affinity, and stability. Moreover, such sensors may be genetically encodable, thus providing

Conversion of a Putative Agrobacterium Sugar-binding Protein into a FRET Sensor with High Selectivity for Sucrose

“…To enhance the sensitivity of the sucrose-selective nanosensor FLIPsuc-90, linker deletions were generated using the same rationale used for improving the glucose nanosensors. Deletion of the composite linkers, which consist of the artificially introduced flexible linker sequences as well as poorly structured fluorophore, should lead to tighter allosteric coupling by reducing rotational averaging (20). FLIPsuc-90⌬1, in which 13 amino acids have been deleted, shows an almost 4-fold increase in signal change.…”

“…Constructs with shortened linkers between the green fluorescent protein variants and ThuE were created from FLIPsuc-90. FLIPsuc-90⌬1 was created by sub-cloning AtThuE into FLIPglu-600⌬5 (20) with KpnI, which created a construct with 13 amino acids deleted from the linkers. FLIPsuc-90⌬2 was created by shortening the linkers to the same length as in FLIPglu-600⌬13, yielding a nanosensor with linkers 25 amino acids shorter (20).…”

“…FLIPsuc-90⌬1 was created by sub-cloning AtThuE into FLIPglu-600⌬5 (20) with KpnI, which created a construct with 13 amino acids deleted from the linkers. FLIPsuc-90⌬2 was created by shortening the linkers to the same length as in FLIPglu-600⌬13, yielding a nanosensor with linkers 25 amino acids shorter (20). FLIPsuc proteins were extracted from BL21(DE3)Gold and purified as described (8).…”

“…Point mutations and single amino acid deletions in the binding protein-fluorophore linker sequences can alter both the sign and magnitude of the signal change (20); it thus appears that more subtle effects than the macroscopic change in fluorophore separation contribute to the observed signal change.…”

“…Linker shortening has proven to be a reliable method for signal enhancement in a variety of nanosensor backgrounds (20). Two sets of amino acid deletions from the Nand C-terminal composite linkers connecting the binding protein with eCFP and eYFP (the composite linker includes poorly structured chromophore regions as well as any explicit linker sequence) were generated; they are a conservative deletion of 13 residues to create FLIPsuc-90⌬1 and a more extensive deletion of 25 residues to create FLIPsuc-90⌬2.…”