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Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

2001; Springer Nature; Volume: 20; Issue: 21 Linguagem: Inglês

10.1093/emboj/20.21.5853

1460-2075

Henrik Østergaard, A. Henriksen, Flemming Hansen, Jakob R. Winther,

Electron Spin Resonance Studies

Article1 November 2001free access Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein Henrik Østergaard Henrik Østergaard Section of Molecular Microbiology, BioCentrum-DTU, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Valby, Denmark Search for more papers by this author Anette Henriksen Anette Henriksen Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Valby, Denmark Search for more papers by this author Flemming G. Hansen Flemming G. Hansen Section of Molecular Microbiology, BioCentrum-DTU, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark Search for more papers by this author Jakob R. Winther Corresponding Author Jakob R. Winther Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Valby, Denmark Search for more papers by this author Henrik Østergaard Henrik Østergaard Section of Molecular Microbiology, BioCentrum-DTU, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Valby, Denmark Search for more papers by this author Anette Henriksen Anette Henriksen Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Valby, Denmark Search for more papers by this author Flemming G. Hansen Flemming G. Hansen Section of Molecular Microbiology, BioCentrum-DTU, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark Search for more papers by this author Jakob R. Winther Corresponding Author Jakob R. Winther Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Valby, Denmark Search for more papers by this author Author Information Henrik Østergaard1,2, Anette Henriksen3, Flemming G. Hansen1 and Jakob R. Winther 2 1Section of Molecular Microbiology, BioCentrum-DTU, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark 2Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Valby, Denmark 3Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Valby, Denmark *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:5853-5862https://doi.org/10.1093/emboj/20.21.5853 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements. The 1.5 Å crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the β-barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway. Introduction While disulfide bonds serve as important structural components of many of the proteins exported from the cell, they are almost completely absent from their cytosolic counterparts (Gilbert, 1990). In the periplasm of Escherichia coli, disulfide bonds are introduced into nascent proteins through the action of the DsbA and DsbC pathways (reviewed by Rietsch and Beckwith, 1998; Debarbieux and Beckwith, 1999). In the cytosol, protein disulfide bonds are also found; however, here they are only formed transiently. Thus, disulfides are found as part of the catalytic cycle of enzymes such as ribonucleotide reductase or in redox-regulated proteins such as the chaperone Hsp33 and the transcription factor OxyR (Aberg et al., 1989; Zheng et al., 1998; Jakob et al., 1999). Maintenance of protein sulfhydryls in general as well as recycling of these proteins to the reduced state relies on two partially redundant pathways, one involving thioredoxins (encoded by trxA and trxC) and the other glutaredoxins (encoded by grxA, B and C) (Prinz et al., 1997; Aslund and Beckwith, 1999). These thiol–disulfide oxidoreductases belong to a family of enzymes having in common a pair of redox-active cysteines in a Cys-Xaa-Xaa-Cys motif (Martin, 1995). Cycling of the active site cysteines between the reduced and oxidized state allows them to undergo disulfide exchange reactions with protein substrates or small thiol compounds such as glutathione. Both pathways rely on NADPH as the source of reducing equivalents. In the thioredoxin pathway, they are delivered to thioredoxin by the flavoenzyme thioredoxin reductase (encoded by trxB), while electrons are transferred via glutathione reductase and glutathione to the glutaredoxins (Holmgren, 1989). Characterization of the pathways leading to the formation or reduction of disulfide bonds has relied extensively on the use of naturally occurring proteins as endogenous probes of the cellular thiol–disulfide redox state. These have been selected based on discernible characteristics of their oxidized and reduced state such as enzymatic activity [e.g. alkaline phosphatase (Derman et al., 1993) and β-galactosidase (Bardwell et al., 1991; Jonda et al., 1999)] and electrophoretic mobility (e.g. OmpA and β-lactamase; Bardwell et al., 1991). However, although a variety of such reporters are available in E.coli, only few exist in yeast and mammalian cells and none support non-invasive redox monitoring at the single-cell level. To overcome these limitations, we have engineered a new type of redox reporter based on the fluorescent properties of green fluorescent protein (GFP). Besides its bright and visible fluorescence, GFP exhibits a number of attractive features for in vivo reporter applications. It is chemically inert and does not interfere with cellular processes; contains no disulfide bonds, and can be targeted specifically to subcellular compartments such as the endoplasmic reticulum, mitochondria and the periplasmic space in bacteria (De Giorgi et al., 1999; Casey et al., 2000). GFP consists of 238 amino acids folded into an 11-stranded β-barrel wrapped around a central irregular α-helix (see Figure 1A) (Ormo et al., 1996; Yang et al., 1996). The chromophore is situated in the middle of the α-helix and is generated by an autocatalytic cyclization of the tripeptide segment -Ser65-Tyr66-Gly67- (Cody et al., 1993; Heim et al., 1994; Reid and Flynn, 1997). Mutagenesis of the chromophore sequence and the surrounding barrel structure has resulted in variants with significantly altered absorption and emission spectra (reviewed by Tsien, 1998; Palm and Wlodawer, 1999; Remington, 2000). One example is the yellow fluorescent variant of GFP (YFP) with a Thr→Tyr mutation at position 203. In the crystal structure of YFP (termed wtYFP in the following), the highly polarizable tyrosine side chain stacks with the phenolic moiety of chromophore, leading to a red shift of the fluorescence spectrum (Wachter et al., 1998). Figure 1.Cysteine mutants of the yellow fluorescent protein. (A) The six positions at which cysteines residues were introduced are marked with black spheres in the crystal structure of wtYFP (Wachter et al., 1998). The chromophore and the side chain of Tyr203 are shown as stick representations. Relevant β-strands are numbered in white according to Yang et al. (1996). (B) Spontaneous oxidation of the cysteine mutants as monitored by non-reducing SDS–PAGE. Reduction of the four mutant proteins was performed by overnight incubation with 20 mM DTT. Subsequently they were dialysed against 20 mM Tris–HCl pH 8.0 for 17 h at room temperature to promote oxidation. Trichloroacetic acid (10% v/v) was added to aliquots of the samples taken before (marked Red) and after (marked Ox) dialysis. The protein precipitate was washed twice with acetone and then alkylated with 50 mM N-ethylmaleimide before analysis by non-reducing SDS–PAGE on a 16% gel. Download figure Download PowerPoint Using YFP as a template, we present here the design and the biochemical and structural characterization of a novel fluorescent disulfide bond reporter and show that it is a sensitive probe of the redox state in the cytoplasm of E.coli. Results Engineering disulfide bonds in YFP The spectral properties of GFPs are determined in part by a number of non-covalent interactions between the chromophore and residues in the surrounding barrel structure. The majority of these residues are situated on the inner side of three consecutive β-strands 7, 10 and 11, shielding the chromophore from the bulk solvent (Figure 1A). In wtYFP, these residues constitute His148, Tyr203, Glu222, Asn146 and Ser205 (Wachter et al., 1998). Previous studies have identified this region of the β-barrel as being structurally flexible, tolerating the introduction of new N- and C-termini by circular permutation and even insertion of entire protein domains (Baird et al., 1999; Topell et al., 1999). This led us to speculate that reversible formation of a strained disulfide in this part of the protein might perturb the surrounding structure sufficiently to bring about a measurable change in the fluorescent properties of the protein. By visual inspection of the crystal structure of wtYFP, four pairs of residues 147–204, 149–202, 202–225 and 204–223, were found suitable for disulfide engineering. As shown in Figure 1A, each pair bridges two of the three β-strands interacting with the chromophore and are, furthermore, solvent exposed to enable free access of the surrounding redox buffer. GFP contains two native cysteines, Cys48 and Cys70. Previous work by Inouye and Tsuji (1994) indicated that Cys48, being partially solvent exposed, might be susceptible to modification by thiol-specific reagents. To avoid interfering side reactions, it was substituted for a valine (see Materials and methods for details). The four proteins were produced in high yield in E.coli, indicating that the mutations did not significantly impair protein folding or autocatalytic formation of the chromophore. In the following, they will be referred to as rxYFPXaa1Xaa2, where Xaa1 and Xaa2 designate the two positions at which cysteines were introduced. To investigate whether the four cysteine variants were able to form disulfide bonds under oxidizing conditions, the purified proteins were dialysed against air-saturated buffer at pH 8.0. Pre-treatment with dithiothreitol (DTT) was performed to reduce any disulfide bonds present in the starting material. For each of the proteins, complete oxidation was found to occur within hours, observed by non-reducing SDS–PAGE as a slight increase in electrophoretic mobility (Figure 1B). One of the mutant forms, rxYFP204223, in addition to forming oxidized monomers, also associated into disulfide-bonded dimers (apparent as a band at ∼60 kDa). These were highly stable and required prolonged incubation with high concentrations of DTT to dissociate (data not shown). Consequently, rxYFP204223 was not investigated further. The most pronounced difference in the fluorescent properties of the reduced and oxidized state was observed for rxYFP149202, where formation of the disulfide resulted in a 2.2-fold reduction of the emission peak at pH 7.0 (Figure 2). In comparison, rxYFP147204 and rxYFP202225 only displayed an ∼1.2-fold change. Apart from the change in amplitude, the spectra of the two redox states were essentially identical and similar to those of wtYFP, with excitation and emission peaks at 512 and 523 nm, respectively, and a minor shoulder around 488 nm. The substantial spectral response of rxYFP149202 to changes in its redox state prompted us to characterize this variant in further detail. Figure 2.Fluorescence excitation and emission spectra of oxidized (dashed line) and reduced (solid line) rxYFP149202 in 100 mM potassium phosphate pH 7.0, 1 mM EDTA at 30°C. The two spectra were recorded at emission and excitation wavelengths of 540 and 490 nm, respectively. The fluorescent properties of reduced rxYFP149202 are identical to those of the template YFP used (see Materials and methods). Download figure Download PowerPoint Stability and reactivity of the C149–C202 disulfide bond The redox potential of the cysteine couple in rxYFP149202 was determined from the equilibrium constant of the reaction with glutathione at pH 7.0 and 30°C. By measuring the intrinsic fluorescence of rxYFP149202 equilibrated in buffers with various ratios of reduced and oxidized glutathione (abbreviated GSH and GSSG, respectively), Kox was determined to 5.0 ± 0.15 M (Figure 3A). Analysis of the equilibrium mixtures by non-reducing SDS–PAGE (Figure 3A, inset) yielded the same value of Kox and thereby reconfirmed the tight correlation between the intrinsic fluorescence of the protein and its redox state. For the calculation of Kox, it was assumed that the concentration of protein–glutathione mixed disulfide at equilibrium was insignificant, an assumption that was justified by the close fit of the model to the experimental data as well as the high effective molarity of the cysteine residues relative to the concentrations of glutathione used (the equilibrium constant for protein–glutathione mixed disulfides are generally 104 molar excess of DTT (Figure 3B). From a fit of the progress curves to a single exponential function, the apparent second-order rate constant (k2) was estimated to 24.8 ± 0.6 M/min, which is close to the value of 14.1 M/min reported for reduction of oxidized glutathione by DTT (Szajewski and Whitesides, 1980). Spectral properties of rxYFP149202 Next, we investigated the mechanism underlying the redox state-dependent shift in fluorescence emission. Figure 4A shows the absorption spectra of the protein at five different points on the redox titration curve given in Figure 3A. Peaks are observed at 404 and 512 nm. Previous studies have shown the two absorption bands to arise from a protonated and deprotonated state of the chromophore, respectively (Chattoraj et al., 1996; Niwa et al., 1996; Brejc et al., 1997; Elsliger et al., 1999). The phenolic moiety of Tyr66 is responsible for this behaviour (Elsliger et al., 1999); it is a weak monoprotic acid and, despite its location in the interior of the β-barrel, it responds rapidly to changes in the external pH. In YFP, the protonated chromophore is non-fluorescent. The increase in the 404/512 nm peak ratio upon oxidation suggests that bridging of the two cysteines shifts the equilibrium between the two chromophore protonation states towards the non-fluorescent, protonated form. This was confirmed by fluorescence pH titrations of the free and disulfide-bonded protein yielding apparent chromophore pKa values of 6.05 ± 0.01 [Hill coefficient (n) = 1.00] and 6.76 ± 0.01 (n = 0.86), respectively (Figure 4B). Similar values, 6.00 ± 0.01 (n = 0.98) and 6.70 ± 0.03 (n = 0.79), were obtained by absorbance measurements. A shift in the chromophore ionization constant of 0.7 units alone, however, is not sufficient to account for the observed change in the intrinsic fluorescence. Disulfide bond formation also leads to an ∼1.5-fold reduction in the molar extinction coefficient of the deprotonated chromophore, which is evident from the lack of convergence of the two absorption pH titration curves at high pH, where the chromophore is predominantly negatively charged (see Figure 4B). Figure 4.Redox state-dependent spectral properties of rxYFP149202 at 30°C. (A) Absorption spectra were recorded after equilibration of the protein in redox buffers with [GSH]2/[GSSG] ratios of 0.004 M (open circles), 0.55 M (open squares), 2.17 M (open diamonds) and 8.13 M (open triangles) (see Figure 3A). Reduced protein (open inverted triangles) was obtained by incubation with 10 mM DTT for 2 h. (B) The relative fluorescence (open symbols) and relative absorbance (closed symbols) of reduced (circles) and oxidized (squares) rxYFP149202 as a function of pH. Data were fitted as described in Materials and methods. The estimated values of the chromophore pKa and the Hill coefficient (n) are given in the text. Download figure Download PowerPoint Crystallographic analysis of oxidized rxYFP149202 To define the structural basis of the redox state-dependent fluorescence, the crystal structure of oxidized rxYFP149202 was determined. The protein was crystallized at pH 8.0 to ensure a homogenous population of deprotonated chromophores. Using wtYFP as a search model, the structure was solved by molecular replacement. The final resolution was 1.5 Å, with R-factors of 18.3 and (Rfree) 21.2%, respectively (data collection and refinement statistics are given in Table I). Table 1. Data collection and refinement statistics Data collection Refinement Wavelength (Å) 1.0362 Rconv (%) 18.3 X-ray source 711 Lund Rfree (%) 21.2 Resolution (Å) Resolution (Å) 78.09−1.50 overall 78.09−1.50 No. of reflections outer shell 1.55−1.50 Working set 109 885 No. of reflections Test set 5546 overall 750 960 No. of atoms unique 116 321 Protein 5627 Mosaicity 0.25 Solvent 840 Rsym (%) B-factor, protein atoms (Å2) 16.8 overall 3.3 B-factor, solvent atoms (Å2) 31.1 outer shell 43.9 R.m.s.d. bond distance (Å) 0.011 I/σI R.m.s.d. bond angle (°) 1.8 overall 18.3 Ramachandran plot, residues in outer shell 2.0 Most favourable regions (%) 90.8 Completeness (%) Additional allowed regions (%) 9.0 overall 89.1 Disallowed regions (%) 0.0 outer shell 72.9 Estimated coordinate error from sigmea (Å) 0.16 C–V estimated coordinate error from sigmea (Å) 0.16 Overall, the crystal structure of rxYFP149202 resembles that of wtYFP, with a root mean square deviation of 0.5 Å for the α carbons (Figure 5A). As shown in Figure 5B, significant 2Fo − Fc electron density is visible between β-strands 7 (residues 147–153) and 10 (residues 199–208) corresponding to the disulfide bridge, which is completely exposed on the surface of the protein. The cysteine side chain adopts a right-handed staple conformation, which is identical to that of naturally occurring disulfides spanning adjacent antiparallel β-strands (Harrison and Sternberg, 1996). Due to the geometrical requirements of the disulfide, the distance separating the two Cα atoms is reduced by 0.8 Å relative to wtYFP, resulting in a local constriction of the two β-strands at the site of the disulfide bridge (Figure 5C). This is brought about mainly by a movement of Cys149 towards β-strand 10. Movement of Asn164, Phe165 and Lys166 (part of β-strand 8) in the opposite direction leads to an unzipping of the β-sheet between strands 7 and 8 and the concomitant loss of three main chain hydrogen bonds (one of them mediated by a water molecule; marked with dashed lines in Figure 5C). On the molecular surface, the strand separation is evident as a narrow, water-filled groove running along the β-strands. However, direct solvent access to the chromophore, which is located immediately behind the strands, is prevented due to steric blocking by the side chain of His148 (see below). Figure 5.Crystal structure of oxidized rxYFP149202. (A) Superposition of oxidized rxYFP149202 (throughout the figure in blue) and wtYFP (throughout in yellow; Wachter et al., 1998) (r.m.s. deviation is 0.5 Å for equivalent Cα atoms). The chromophore, Cys149 and Cys202 are shown as ball-and-stick representations with the disulfide bond coloured green. (B) The 2Fo − Fc electron density map of the C149–C202 disulfide bridge (contoured at 1σ in blue and at 8σ in green). Superimposed is the final model of the disulfide bridge, which adopts the right-handed staple conformation. (C) Overlay of the backbone trace of wtYFP and oxidized rxYFP149202. Also shown are the disulfide bond (in green), the side chain of His148 and the chromophore (Cro66). The dashed lines indicate those hydrogen bonds that are present in wtYFP but absent in the rxYFP149202 structure. (D) Position of Tyr203 and His148 relative to the chromophore. The superimposed chromophore structures of rxYFP149202 and wtYFP are shown in grey. Dashed lines indicate hydrogen bonds to the δ1 and ϵ2 nitrogens of His148. Download figure Download PowerPoint In wtYFP, the imidazole of His148 is wedged between strands 7 and 8 (Wachter et al., 1998). It receives a hydrogen bond from the backbone of Arg168 and serves as an obligate hydrogen bond donor, via Nδ1, to the phenolate oxygen of Tyr66 (Figures 5C and 6D). Site-directed mutagenesis of His148 has shown this interaction to be highly important in stabilizing the negatively charged deprotonated state of the chromophore (Elsliger et al., 1999). Substitution by glycine or glutamine residues, which do not hydrogen-bind to the chromophore, lowers the equilibrium constant for ionization by more than one order of magnitude (Wachter et al., 1998, 2000). In oxidized rxYFP149202, a major repositioning of His148 has taken place. Relative to wtYFP, it has moved away from the plane of the β-sheet by 1.4 Å and the imidazole side chain has rotated 108° around the χ1 bond (Figure 5D). It is now in a position where it can interact with the chromophore phenolic oxygen through the Nϵ2 atom. As the hydrogen bond partner to the Nδ1 atom is a solvent molecule (located in the groove between the β-strands 7 and 8), Nϵ2 can serve as both a donor and an acceptor of the hydrogen bond depending on the protonation state of Tyr66. The loss of directionality of the hydrogen bond is most likely to be the major cause of the observed increase in the chromophore pKa. Figure 6.Redox state of rxYFP149202 expressed in wild-type E.coli and a trxB mutant at 30°C. (A) Fluorescence was monitored continuously at 523 nm in a standard fluorimeter (see Materials and methods). From the initial fluorescence and the fluorescence after sequential addition of 50 μl of 3.6 mM 4-DPS and 50 μl of 1 M DTT to 900 μl of the cultures, the proportion of oxidized reporter in the two strains was calculated to 53% (wild type) and 86% (trxB), respectively. (B) Western blot detection of rxYFP149202 in the cultures before (lane 1) and after treatment with 4-DPS and DTT (lanes 2 and 3, respectively). In the lanes marked ‘Control’ were loaded equal amounts of reduced and oxidized rxYFP149202 at increasing dilution to show that the anti-GFP antibodies used bind with lower affinity to the oxidized reporter. Quantitative densitometric analysis of the bands corresponding to the untreated cultures, taking into account the weaker binding of the antibodies to the oxidized protein, indicated 50% oxidized reporter in the wild type and 83% in the trxB mutant. Download figure Download PowerPoint In the interior of the protein, the chromophore has moved 0.7 Å away from the gap in the β-sheet and now occupies a position similar to that of wild-type GFP and S65T GFP (Ormo et al., 1996; Yang et al., 1996). The side chain of Tyr203 has rotated 30° around the χ1 bond in the same direction. Compared with the off-centred parallel displaced arrangement of the Tyr203 side chain and the chromophore phenol in wtYFP, the two aromatic rings are positioned at skewed angles in rxYFP149202 (Figure 5D). It presently is not clear to what extent this misalignment or other small-scale movements of side chains in the immediate chromophore environment contribute to the observed drop in the molar extinction coefficient upon oxidation. Redox monitoring in the cytoplasm To demonstrate the functionality of rxYFP149202 in vivo, it was expressed in the cytoplasm of wild-type E.coli and an isogenic strain disrupted in the trxB gene encoding thioredoxin reductase. Deficiency of trxB leads to the accumulation of thioredoxin in the oxidized state, which promotes the incorporation of disulfide bonds into nascent cytoplasmic proteins (Derman et al., 1993; Stewart et al., 1998). Fluorescence measurements were carried out in a standard fluorimeter after expression of the reporter from the lac promoter in exponentially growing cells for >10 generations. As expected, fluorescence intensity per OD600 was found to be highest for the wild type, indicating increased levels of oxidized rxYFP149202 in the trxB mutant (Figure 6A). Due to the dependency of fluorescence intensity on the protein concentration, calibration was required to determine the exact ratio between oxidized and reduced reporter. This was carried out by measuring the fluorescence after sequential addition of oxidant (Fox) and reductant (Fred) directly to the culture in the cuvette. After addition of the membrane-permeable thiol-oxidant 4,4′-dithiodipyridine (4-DPS; Grassetti and Murray, 1967) and DTT, the fraction of oxidized reporter in the wild type and the trxB mutant was found to be 53 ± 3 and 86 ± 3%, respectively. Similar distributions were obtained by western blotting (Figure 6B). Oxidation of rxYFP149202 also occurred after addition of the widely used thiol-oxidant diamide although with a rate constant more than two orders of magnitude lower than that of oxidation by 4-DPS. Based on the measured values of Fred/Fox and the correlation between fluorescence and pH given in Figure 4B, the pH in the cytoplasmic compartment of the two strains could be estimated to 7.4 ± 0.1. This corresponds well with the value of 7.5 determined by 31P NMR in aerobically respiring E.coli cells grown at neutral pH (Slonczewski et al., 1981). While oxidation of the reporter by 4-DPS was too fast (within the mixing time in the cuvette) to allow for a kinetic characterization of the reaction, reduction by DTT occurred over minutes. The progress curve conformed to pseudo first-order kinetics from which apparent second-order rate constants of 30 ± 1 M/min (wild type) and 28 ± 1 M/min (trxB mutant) could be extracted. The elevated pH of the cytosol most probably accounts for the slightly higher values of k2 as compared with those determined in vitro (see Figure 3B). Discussion Functional and structural properties of rxYFP149202 In an effort to visualize the formation of disulfide bonds in living cells, redox-active cysteines were introduced into YFP. Of the four double-cysteine mutants constructed, only rxYFP149202 exhibited a change in the intrinsic fluorescence sufficient to enable in vivo redox monitoring. The >2-fold change observed at neutral pH is comparable in magnitude with the dynamic range of other YFP-based reporters (Miyawaki et al., 1997; Jayaraman et al., 2000). However, due to differential pH sensitivities of the reduced and oxidized state, the dynamic range was found to vary substantially according to the pH. Thus, at pH 6.5, correspond