Development of roGFP2-derived redox probes for measurement of the glutathione redox potential in the cytosol of severely glutathione-deficient rml1 seedlings

Abstract

Glutathione is important for detoxification, as a cofactor in biochemical reactions and as a thiol-redox buffer. The cytosolic glutathione buffer is normally highly reduced with glutathione redox potentials (E GSH ) of more negative than -310 mV. Maintenance of such negative redox potential is achieved through continuous reduction of glutathione disulfide by glutathione reductase (GR). Deviations from steady state glutathione redox homeostasis have been discussed as a possible mean to alter the activity of redox-sensitive proteins through switching of critical thiol residues. To better understand such signaling mechanisms it is essential to be able to measure E GSH over a wide range from highly negative redox potentials down to potentials found in mutants that show already severe phenotypes. With the advent of redox-sensitive GFPs (roGFPs), understanding the in vivo dynamics of the thiol-based redox buffer system became within reach. The original roGFP versions, roGFP1 and roGFP2, however, have midpoint potentials between -280 and -290 mV rendering them fully oxidized in the ER and almost fully reduced in the cytosol, plastids, mitochondria, and peroxisomes. To extend the range of suitable probes we have engineered a roGFP2 derivative, roGFP2-iL, with a midpoint potential of about -238 mV. This value is within the range of redox potentials reported for homologous roGFP1-iX probes, albeit with different excitation properties. To allow rapid and specific equilibration with the glutathione pool, fusion constructs with human glutaredoxin 1 (GRX1) were generated and characterized in vitro. GRX1-roGFP2-iL proved to be suitable for in vivo redox potential measurements and extends the range of E GSH values that can be measured in vivo with roGFP2-based probes from about -320 mV for GRX1-roGFP2 down to about -210 mV for GRX1-roGFP2-iL. Using both probes in the cytosol of severely glutathione-deficient rml1 seedlings revealed an E GSH of about -260 mV in this mutant.

Keywords: GRX1-roGFP2; glutathione; glutathione redox potential; redox imaging; rml1.

Figure 1

2D models of roGFP2 and roGFP2-iL illustrate the relative position of disulfide forming Cys147 and Cys204. (A) Cys147 and Cys204 are located on strands 7 and 10, respectively, and are positioned in close proximity so that they can facilitate disulfide formation. (B) The introduction of a leucine next to Cys147 (Leu147^*) and the substitution of His148 by serine increase the relative distance between Cys147 and Cys204 that causes alterations in the thermodynamic stability of the disulfide. (C) Schematic representation of the GRX1-roGFP2-iL fusion protein. GRX1 was fused to the N-terminus of roGFP2-iL by a TS(GGSGG)₆ linker.

Figure 2

Excitation properties of roGFP2 and roGFP2-iL. Excitation spectra of roGFP2 (A) and roGFP2-iL (B) in fully oxidized (red curve) and fully reduced (blue curve) state. Emission was monitored at 540 nm. The maximum dynamic ranges (δ) were calculated from the 405/488 nm excitation ratios for fully reduced and fully oxidized probes. Both excitation wavelengths are indicated by vertical lines.

Figure 3

pH-dependence of roGFP2-iL fluorescence. Recombinant roGFP2-iL was incubated in potassium-phosphate buffer at different pH values. The changes in roGFP2-iL fluorescence intensity of the 390 nm (black) and 480 nm channel (white) in buffers of different pH values under full oxidation with 10 mM H₂O₂ (A) and full reduction by 10 mM DTT (B) is shown. Increasing pH values beyond pH 7.0 equally increase fluorescence intensity in both channels thus the 390/480 nm ratio remains unaffected. Conversely, decreasing pH below pH 7.0 has the opposite effect for fully oxidized and fully reduced roGFP2-iL. Similar pH-dependent effects have been reported for wild-type GFP (Patterson et al., 1997). (C) The excitation ratios (390/480 nm) in 10 mM H₂O₂ (♦) and in 10 mM DTT (■) of roGFP2-iL are plotted against the respective pH solution. For several data points, standard error is smaller than data marker (n = 3 technical replicates).

Figure 4

Redox titration of roGFP2-iL and GRX1-roGFP2-iL. The degree of reduction of roGFP2-iL (A) and GRX1-roGFP2-iL (B) was determined from the excitation ratios plotted against the potential of the ambient redox buffer. Representative measurements of at least 3 independent redox titrations (with 4 replicates each) for both probes are shown.

Figure 5

Redox titration of roGFP2 and GRX1-roGFP2. The degree of reduction of roGFP2 (A) and GRX1-roGFP2 (B) was determined from the excitation ratios plotted against the potential of the ambient redox buffer. Representative measurements of at least 3 independent redox titrations (with 4 replicates each) for both probes are shown.

Figure 6

The N-terminally fused glutaredoxin catalyzes the transfer of electrons from glutathione to roGFP2-iL. The excitation ratio 390/480 nm with emission at 520 nm was followed over time. Fully reduced glutathione was added to a solution of oxidized roGFP2-iL () or GRX1-roGFP2-iL (), respectively 2.5 min after the start of the measurement. Change of reduction was recorded over time after injection of 2 mM reduced glutathione. The reaction buffer contained 100 μM NADPH and glutathione reductase at pH 7.4 to maintain full reduction of glutathione over the measured time (n = 4).

Figure 7

GSH-dependent reduction of roGFP variants. The kinetics for the reduction of roGFP by reduced glutathione strongly depends on redox potential of the disulfide forming cysteines of the respective roGFP sensors. (A) The excitation ratio 390/480 nm with emission at 520 nm was followed over time. Fully reduced glutathione (final concentration 5 mM) was added to a solution of pre-oxidized roGFP1-iL (), roGFP2-iL (), roGFP2 (), and roGFP1 () 3.75 min after start of the measurement. The reaction buffer contained 2 μM GRX, 100 μM NADPH, and glutathione reductase at pH 7.4 to maintain maximum reduction of glutathione over the measured time course (n = 4). (B) dRed/dt further illustrates the rapid change in reduction of roGFP1-iL () and roGFP2-iL () compared to roGFP2 () and roGFP1 ().

Figure 8

The GRX1-roGFP2-iL probe expressed in the cytosol of homozygous rml1 mutants is almost completely reduced while GRX1-roGFP2 is highly oxidized. (A) Ratiometric images (a–f) show the redox state of GRX1-roGFP2 and GRX-roGFP2-iL in the cytosol of root epidermal cells of rml1 mutants. Treatment with 10 mM DTT results in full reduction of GRX1-roGFP2 (a) and GRX-roGFP2-iL (d), while under resting conditions GRX1-roGFP2 is highly oxidized (b) whereas GRX1-roGFP2-iL is in a reduced state (e). Treatment with 25 mM H₂O₂ results in complete oxidation of GRX1-roGFP2 (c) and GRX-roGFP2-iL (f). Scale bar 10 μm. (B,C) Fluorescence ratio of cytosolic GRX1-roGFP2 (B) and GRX1-roGFP2-iL (C) show complete reduction and oxidation of GRX-roGFP2 and GRX1-roGFP2-iL after treatment with 10 mM DTT and 25 mM H₂O₂, respectively. Under resting conditions in H₂O, GRX1-roGFP2 is highly oxidized while GRX1-roGFP2-iL is highly reduced. Values are calculated as the mean ± SD (n = 5 − 10).

Figure 9

Deduction of the redox potential of the roGFP probes from the Degree of Oxidation (OxD) in the cytosol of rml1 seedlings. (A) GRX1-roGFP2-iL and GRX1-roGFP2 expressed in the cytosol of rml1 plants as calculated according to equations given in Materials and Methods from the mean fluorescence data presented in Figure 7. While GRX1-roGFP2 is highly oxidized only a small fraction of GRX1-roGFP2-iL is in the oxidized state. (B) Titration curves drawn for GRX1-roGFP2 (blue) and GRX1-roGFP2-iL (red) were calculated from the Nernst-Equation using the midpoint potentials determined in Figures 3, 4. Dotted horizontal lines refer to OxD of GRX1-roGFP2 and GRX1-roGFP2-iL from rml1 measurements shown in panel (A). The vertical dotted line indicates the interception points for GRX1-roGFP2 (blue) and GRX-roGFP2-iL (red) which suggests an E_GSH of about −260 mV in the cytosol of rml1.