Single-fluorophore membrane transport activity sensors with dual-emission read-out

Abstract

We recently described a series of genetically encoded, single-fluorophore-based sensors, termed AmTrac and MepTrac, which monitor membrane transporter activity in vivo (De Michele et al., 2013). However, being intensiometric, AmTrac and Meptrac are limited in their use for quantitative studies. Here, we characterized the photophysical properties (steady-state and time-resolved fluorescence spectroscopy as well as anisotropy decay analysis) of different AmTrac sensors with diverging fluorescence properties in order to generate improved, ratiometric sensors. By replacing key amino acid residues in AmTrac we constructed a set of dual-emission AmTrac sensors named deAmTracs. deAmTracs show opposing changes of blue and green emission with almost doubled emission ratio upon ammonium addition. The response ratio of the deAmTracs correlated with transport activity in mutants with altered capacity. Our results suggest that partial disruption of distance-dependent excited-state proton transfer is important for the successful generation of single-fluorophore-based dual-emission sensors.

Keywords: S. cerevisiae; biophysics; biosensor; cell biology; excited state proton transfer (ESPT); fluorescence; green fluorescent protein; structural biology; transporter.

Figure 1.. Steady-state and time-resolved fluorescence analysis of AmTrac sensors.

(A) Scheme of AmTrac sensors showing left (LL) and right linker (RL) positions (top). Table indicates amino acid composition of linkers in AmTrac sensors. (B) Normalized fluorescence spectra, excitation (dashed lines; λ_em 514 nm), and emission (solid line; λ_exc 485 nm). Traces in grey represent blank (background fluorescence of untransformed yeast). Values relate to major peak of AmTrac-GS (n = 3). (C) Time-resolved fluorescence decay kinetics of AmTrac-GS and -LE in presence and absence of 1 mM NH₄Cl (λ_exc 475 nm; λ_em 514 nm; n = 3). (D) Time-resolved fluorescence anisotropy r(t) of AmTrac-GS and -LE in presence and absence of 1 mM NH₄Cl (λ_exc 475 nm; λ_em 514 nm). DOI: http://dx.doi.org/10.7554/eLife.07113.002

Figure 1—figure supplement 1.. Summary of lifetime components (τ) and χ² values obtained for AmTrac-GS and -LE treated with water as control or 1 mM NH₄Cl.

In case of multiphasic lifetime components, two values are listed. Data were fitted single- and bi-exponentially (n = 3). DOI: http://dx.doi.org/10.7554/eLife.07113.003

Figure 1—figure supplement 2.. Time-resolved fluorescence decay kinetics of AmTrac-LE in presence and absence of 1 mM NH₄Cl (λ_exc 475 nm, λ_em 514 nm).

The according bi-exponential fit curves are shown in green and blue, respectively. DOI: http://dx.doi.org/10.7554/eLife.07113.004

Figure 2.. Steady-state fluorescence spectra for excited-state proton transfer (ESPT) analysis of yeast transformed with AmTrac-LE and postulated structural differences.

(A) Fluorescence excitation (dashed line; λ_em 530 nm) and emission (solid line; λ_exc 485 nm) after treatment with NH₄Cl at indicated concentrations. Values were normalized to the major peak of the B-band (n = 3). (B) Fluorescence emission spectrum (λ_exc 395 nm). Values normalized to major maximum of the water-treated control (n = 3). Asterisk (*) indicates iso-emissive point. (C) Chromophore environment of crystalized circularly permuted enhanced green fluorescent protein (cpEGFP, PDB 3EVP). Chromophore is depicted in green; residues involved in ESPT via a buried water molecule in blue, left linker residues in orange and red. Top and bottom illustrations show different rotamers of glutamate Glu¹⁴⁸ with indicated distances of hydrogen bonds to chromophore. DOI: http://dx.doi.org/10.7554/eLife.07113.005

Figure 2—figure supplement 1.. Steady-state fluorescence spectrum for ESPT analysis of intact yeast transformed with AmTrac-GS (n = 3) and postulated structural differences.

(A) Fluorescence emission spectrum with λ_exc 395 nm after treatment with NH₄Cl at the concentrations indicated. Values were normalized to the maximum of the water treatment. (B) Chromophore environment of crystalized cpEGFP (PDB 3EVP). The chromophore is depicted in green, the residues involved in ESPT via a buried water molecule in blue and the left linker residues GS that exchanged the LE linker, in grey. Only one linker orientation can be found, which can theoretically form weak hydrogen bonds with the chromophore (indicated distance of the hydrogen bond to the chromophore). DOI: http://dx.doi.org/10.7554/eLife.07113.006

Figure 3.. Steady-state fluorescence titration analysis of intact yeast transformed with deAmTrac-CP and -FP.

(A) Fluorescence emission spectrum (λ_exc 395 nm) recorded after NH₄Cl addition (concentrations indicated). Values normalized to maximum of water treated control. Asterisks (*) indicate the iso-emissive point. A refers to the protonated A-state, I is the deprotonated intermediate I-state of the chromophore. (B) Titration of the fluorescence response at indicated NH₄Cl concentrations plotted as ratio of the intensity of the A-state /I-state (I_A/I_I) (mean ± SE; n = 5). DOI: http://dx.doi.org/10.7554/eLife.07113.007

Figure 3—figure supplement 1.. Summary of properties of AmTrac sensor variants in response to water or 1 mM NH₄Cl with A-state excition (λ_exc = 395–400 nm).

In case of two maxima, the major peak is underlined. I_A:I_I is the ratio of the emission intensity of the A-state (λ_em 490 nm of blue maximum) vs the intensity of the I-state (λ_em 515 nm of green maximum) (mean ± SE; n ≥ 3). DOI: http://dx.doi.org/10.7554/eLife.07113.008

Figure 3—figure supplement 2.. Chromophore environment of crystalized cpEGFP (PDB 3EVP).

The chromophore is depicted in green, the residues involved in ESPT via a buried water molecule in blue. The modified left linker residues are depicted in magenta and yellow for deAmTrac-CP (A) or magenta and purple for deAmTrac-FP (B). Different rotamers are shown for the residues CP and FP when replacing the LE linker in cpEGFP. DOI: http://dx.doi.org/10.7554/eLife.07113.009

Figure 4.. Response ratio and spectral analysis of mutant deAmTrac-CP and -FP.

(A) Fluorescence response to NH₄Cl (indicated concentrations) plotted as ratio of the intensity of the A-state /I-state (I_A/I_I). Data were normalized to water treated control (=1) (mean ± SE; n = 5). (B) Fluorescence emission spectra with λ_exc 395 nm recorded after treatment with NH₄Cl (concentrations indicated). Values are normalized to the maximum of the water treatment (n = 3). DOI: http://dx.doi.org/10.7554/eLife.07113.010

Figure 4—figure supplement 1.. Yeast growth assay of Δmep1,2,3 strain (31019b) transformed with mutant deAmTracs.

Transformants were grown for 3 days on solid media containing the indicated ammonium concentrations or 1 mM arginine as growth control. Note, the high capacity mutants T464D + A141E grew weaker on high ammonium concentrations, most likely due to ammonium toxicity. DOI: http://dx.doi.org/10.7554/eLife.07113.011

Figure 5.. Suggested mechanistic ESPT model to describe the dual-emission in AmTrac-LE and deAmTracs during high and low ammonium transport activity.

A, protonated A-state of the chromophore; I, deprotonated intermediate state; B, deprotonated B-state. Asterisks (*) indicate the excited state upon illumination with the indicated wavelength. DOI: http://dx.doi.org/10.7554/eLife.07113.012