Costantini L.M. et al. Nat. Commun. 6, 7670 (2015).

Fluorescent proteins are widely used to label intracellular targets, but their performance can be affected by subcellular conditions. The oxidizing environment of the mammalian endoplasmic reticulum is particularly challenging because it promotes disulfide-bond formation between cysteines present in fluorescent proteins. Costantini et al. address this issue by engineering versions of blue, cyan, yellow and green fluorescent proteins that lack native cysteine residues. These changes were not straightforward; for example, cysteine-to-serine substitutions rendered the fluorescent proteins dark. Interestingly, the team found that cysteine-to-valine substitutions, along with additional mutations, led to fluorescent proteins that fold well in the lumen of the endoplasmic reticulum and retain much of the brightness of the parent proteins. These tools will be useful for multicolor studies of proteins in oxidizing environments.