Redox-sensitive green fluorescent protein: Probes for dynamic intracellular redox responses. A review¶

Authors: Mark B. Cannon, S. James Remington

Takeaways:

Abstract¶

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 of intracellular 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.

Main¶

More recently, it has come to light that cysteine reactivity, in particular toward reactive oxygen and nitrogen species (ROS and RNS), is also critical for dynamic processes such as gene regulation, oxidative stress response, and cell signaling (1).

Upon excitation, the neutral chromophore, which is the dominant ground-state form in wild-type GFP, undergoes rapid ESPT from the Tyr66 phenol via a hydrogen bonding network to internal Glu222. The anionic form of the chromophore, which can also be directly excited at 480 nm, then emits a 510-nm light.

Ratiometric sensors are particularly desirable, because they reduce or eliminate measurement errors due to changes in illumination intensity, cell thickness or indicator concentration.

The probes, termed redox-sensitive green fluorescent proteins (roGFPs), were constructed by placing pairs of cysteine residues on neighboring strands on the surface of the GFP β-barrel in positions favorable for formation of disulfide linkages. Two sites were selected: positions 149/202, and positions 147/204.

Half of the roGFPs were developed from wtGFP (roGFP1, roGFP3, and roGFP5) and half from the S65T GFP background (roGFP2, roGFP4, and roGFP6).

A crystal structure of roGFP2 shows that disulfide formation between the pair of engineered surface cysteine residues results in a shift of one β-strand relative to the other (20), which causes subtle internal structural rearrangements, including repositioning of side chains contacting the chromophore (i.e., His148 and Ser205), such that the neutral chromophore is favored over the anionic. Therefore, as a population of roGFP is oxidized, disulfide formation leads to an increase in the excitation peak at 400 nm at the expense of the 480 nm peak (20) (see Fig. 4.4).