Current methods for determining ambient redox potential in cells are labor-intensive and generally require destruction of tissue. This precludes single cell or real time studies of changes in redox poise that result from metabolic processes or environmental influences. By substitution of surface-exposed residues on the Aequorea victoria green fluorescent protein (GFP) with cysteines in appropriate positions to form disulfide bonds, reduction-oxidation-sensitive GFPs (roGFPs) have been created. roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo. Crystal structure analyses of reduced and oxidized crystals of roGFP2 at 2.0-and 1.9-Å resolution, respectively, reveal in the oxidized state a highly strained disulfide and localized main chain structural changes that presumably account for the state-dependent spectral changes. roGFP1 has been targeted to the mitochondria in HeLa cells. Fluorometric measurements on these cells using a fluorescence microscope or in cell suspension using a fluorometer reveal that the roGFP1 probe is in dynamic equilibrium with the mitochondrial redox status and responds to membrane-permeable reductants and oxidants. The roGFP1 probe reports that the matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near ؊360 mV (assuming mitochondrial pH ϳ8.0 at 37°C). In other work (C. T. Dooley, T. M. Dore, G. Hanson, W. C. Jackson, S. J. Remington, and R. Y. Tsien, submitted for publication), it is shown that the cytosol of HeLa cells is also unusually reducing but somewhat less so than the mitochondrial matrix.

Redox-sensitive variants of the green fluorescent protein (roGFPs) had previously been developed that allow "real-time" monitoring of the redox status of cellular compartments by fluorescence excitation ratiometry. However, the response time of these probes limits the study of certain rapid oxidative events, such as H 2 O 2 bursts in cell signaling. The substitution of up to three positively charged amino acids adjacent to the introduced disulfide in roGFP1 (variants designated roGFP1-R1 through -R14) substantially improved the response rate. The pseudo first-order rate constants for oxidation by H 2 O 2 and reduction by DTT and redox midpoint potentials were determined. The rate constants approximately doubled with each additional positively charged substitution, to nearly an order of magnitude total. The midpoint potentials are highly correlated with the rate increases, becoming more oxidizing with increasing numbers of positive substitutions. Crystal structures of two variants with opposite disulfide oxidation states have been determined: a 2.2 Å resolution structure of oxidized "R7" containing two basic substitutions, and a 1.95 Å resolution structure of reduced "R8" with one basic and one acidic substitution. Nonlinear Poisson-Boltzmann (PB) calculations are shown to accurately predict the effects of the substitutions on the rate constants. The effects of the substitutions on dimer formation, relative oxidative midpoint potentials, and oxidation and reduction rates are discussed. roGFPs are demonstrated to constitute an excellent model system for quantitative analysis of factors influencing thiol transfer reactions. roGFP1-R12 is most suitable for use in live cells, due to significantly increased reaction rate and increased pI.Keywords: roGFP; redox; biosensor; disulfide conformation; midpoint potential; active-site design; rate enhancement; electrostatics calculationThe green fluorescent protein from Aequorea victoria has two widely separated excitation maxima whose ratio depends on the structure of the molecule and hence can depend on external conditions (Ormo et al. 1996;Tsien 1998). The two excitation maxima correspond to the neutral and anionic forms of the chromophore (Chattoraj et al. 1996;Brejc et al. 1997), which have absorbance maxima at ,395 and 475 nm, respectively. This feature, which is unique among fluorescent proteins characterized to date, allows the construction of ratiometric indicators that can be targeted to subcellular organelles (Nagai et al. 2001;Hanson et al. 2002). Based on this principle, redox-sensitive probes have been developed . In these indicators, surface-exposed cysteine pairs were introduced onto neighbouring strands of the b-barrel, in positions that allow disulfide formation (roGFP1-6). Structural studies of roGFP2 in the reduced and oxidized states reveal that formation of the Cys147-Cys204 disulfide causes Reprint requests to: S. James Remington, Department of Physics, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229, USA; e-mail: jre...

The quantification of transient redox events within subcellular compartments, such as those involved in certain signal transduction pathways, requires specific probes with high spatial and temporal resolution. Redox-sensitive variants of the green fluorescent protein (roGFP) have recently been developed that allow the noninvasive monitoring ofintracellular thiol-disulfide equilibria. In this chapter, the biophysical properties of these probes are discussed, including recent efforts to enhance their response times. Several recent applications of roGFPs are highlighted, including roGFP expression within Arabidopsis to monitor redox status during root elongation, expression in neurons to measure oxidative stress during ischemia, and targeting of roGFPs to endosomal compartments demonstrating unexpectedly oxidizing potentials within these compartments. Possible future directions for the optimization of roGFPs or new classes of redox-sensitive fluorescent probes are also discussed.

No abstract