optimize a FRET-based biosensor

This post “Optical imaging of neuronal glutamate release and spillover with GluSnFR” from Brain Windows caught my attention. I go to the original and has the following notes about biosensor optimization. The work is from the Roger Tsien’s lab, which represent the most cutting edge procedures. Still, the signal-to-noise ratio is “low” in my opinion. I think it is due to the FRET nature.

Sensor Optimization.
FRET sensor optimization is an area of active research, with numerous recent reports on improved fluorescent proteins, FRET pairs, substrate and linker mutations and screening techniques. Optimization of GluSnFR demonstrates the delicate sensitivity of FRET reporters to their constituents. The fitness landscape of GluSnFR linkers was surprisingly peaked. A single construct, GluSnFR8N5C, of the 176 tested was far superior to all others (Fig. 2c). Although there was general improvement in sensor response as linkers were truncated, the process was nonmonotonic, limiting the predictability of this design strategy. We attempted systemic substitution of fluorescent proteins (FPs) and circularly permuted FPs to improve response, including the fluorescent protein variants ECFP-A206K, Cerulean (27), CyPet, YPet (28), Venus (17) and cpVenus (M145, 157, 173, 195). Unexpectedly, substitutions of improved components to linker-optimized GluSnFRs reduced either the quality of the reporter’s surface expression or response magnitude. This is likely due to linker sequences being already highly tuned for the specific chromophore orientations and subtle electrostatic interactions of the GltI domain and the FP pair.

We inserted ECFP into the putative transmembrane loops of GltI, similar to FLI81PE reported by Deuschle et al. (18). When expressed as a purified protein, FLI81PE has a twofold ratio change to glutamate, but this and all tested ECFP insertion mutants fail to express properly on the surface of mammalian cells. Incorporation of superfolding GFP mutations (29) into the inserted ECFP improved folding and trafficking of many insertion mutants. However, our best-case Rmax when expressed on cell surface was only 4% (data not shown). Further improvements of sensor response may be possible by FP substitution or insertion but will require a rescreening of many linker combinations for that pair.

Screening of single circularly permuted fluorescent protein insertions into GltI may produce a single wavelength glutamate sensor analogous to camgaroos (19). A single-FP GluSnFR could be of practical use in more challenging preparations, such as 2-photon in vivo imaging, when using multiple optical sensors simultaneously or when quantitative calibration is not a priority.

SuperGluSnFR represents a major improvement over other optical indicators of glutamate. No previous membrane-tethered PBP-based FRET reporter has achieved more than a 10% FRET ratio change for glutamate or any other substrate. Another recently reported glutamate sensor, EOS (30), requires purification of recombinant protein followed by thiol-mediated dye labeling and cell surface immobilization through biotinylation. The apparent glutamate off-rate of EOS is on the order of hundreds of milliseconds. In contrast, SuperGluSnFR is genetically expressed, allowing cell-specific or subcellular targeting, has been quantitatively calibrated, and has adequate response size, sensitivity and kinetic rates to resolve single action potentials. Future versions of SuperGluSnFR may be genetically targeted to the active zone by fusion to specific synaptic proteins or targeting motifs, raising the possibility of direct comparison of synaptic vs. extrasynaptic glutamate dynamics. For synaptic targeting, a GluSnFR variant with lower glutamate affinity, such as R25K or E26, would be desired to prevent sensor saturation
in the synaptic cleft.

Library Construction and Screening.
Affinity mutations were made in GltI-pRSETB, using QuikChange (Qiagen); transferred to GluSnFR0N0C-pRSETB by digestion and ligation at the SphI and SacI sites; and assayed in vitro as above. GltI(S73T) was incorporated into GluSnFR0N0C-pDisplay by digestion and ligation at SphI and SacI sites. Glutamate affinity was assayed by bath changes of HBSS with increasing [glutamate] on transfected HEK293 and HeLa cells.

Preliminary large truncations of the N and C termini of GltI indicated that deletions beyond the first putative -helix element of the N and C termini (data not shown) caused misfolding. Therefore, the library was limited to 176 combinations of deletions of 0 –15 aa of the N terminus and 0 –10 of the C terminus of GltI. Truncation combinations were amplified with Phusion polymerase (NEB) and purified with 96-well PCR cleanup cartridges (Qiagen). Truncations were digested with SphI and SacI, ligated into the GSFR0N0C-pDisplay vector, replacing the full-length GltI domain, and plated on selective media. Two colonies of each transformation were cultured and miniprepped. Proper insert length was checked for all by analytical restriction digests.

HEK293 or HeLa cells were seeded on 96-well culture plates, grown for one day, and transfected with one of the 176 2 minipreps. Two days after transfection, glutamate responses we measured by thorough bath exchanges with 0 and 100 M [glutamate] HBSS. Repeated optical measurements were made on selected fields in each well with a 20 air objective and a motorized stage. A random library sample and all large responders were sequenced. 0N0C, 8N0C and 8N5C truncations underwent confirmatory imaging in transfected HEK293 cells on 12-mm coverslips in a Warner imaging chamber with a 40 oil-immersion objective.