Researchers from Osaka University have developed new green fluorescent protein (GFP) that could withstand low pH environment for imaging of acidic organelles. The findings were published in Cell Chemical Biology.

Visualizing cellular components and processes at the molecular level is important for understanding the basis of any biological activity. Fluorescent proteins (FPs) are one of the most useful tools for investigating intracellular molecular dynamics.

However, FPs have usage limitations for imaging in low pH environments, such as in acidic organelles, including endosomes, lysosomes, and plant vacuoles. In environments of pH less than 6, most FPs lose their brightness and stability due to their neutral pKa. pKa is the measure of acid strength; the smaller the pKa is, the more acidic the substance is.

Hajime Shinoda, lead author of the study from Osaka University said even though there are reports of several acid-tolerant green FPs (GFPs), most have serious drawbacks. Furthermore, there is a lack of acid-tolerant GFPs that are practically applicable to bioimaging, he added.

The study aimed to design acid-tolerant monomeric GFP that is practically applicable to live-cell imaging in acidic organelles. In the current study, the team developed an acid-tolerant GFP, called it Gamillus.

Gamillus is a GFP cloned from Olindias formosa (flower hat jellyfish) and exhibits superior acid tolerance (pKa=3.4) and nearly twice as much brightness compared with the reported GFPs. The fluorescence spectrum is constant between pH4.5 and 9.0, which falls between the intracellular ranges in most cell types.

X-ray crystallography (a technique used for determining the atomic and molecular structure of a crystal, in this case, a Gamillus crystal) and point mutagenesis suggest the acid tolerance of Gamillus is attributed to stabilization of deprotonation in its chemical structure.

The corresponding author of the study, Takeharu Nagai said: "The applicability of Gamillus as a molecular tag was shown by the correct localization pattern of Gamillus fusions in a variety of cellular structures, including ones that are difficult to target. We believe Gamillus can be a powerful molecular tool for investigating unknown biological phenomena involving acidic organelles, such as autophagy."