AsianScientist (Jan. 10, 2018) – A team of scientists at Osaka University, Japan, has developed a protein-based fluorescent probe that is acid tolerant, allowing the visualization of specific structures in biological cells. They published their findings in the journal 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.
In this study, a research group led by Dr. Hajime Shinoda of Osaka University developed an acid-tolerant green fluorescent protein (GFP), cloned from the flower hat jellyfish Olindias formosa.
“There is a lack of acid-tolerant GFPs that are practical for use in bioimaging,” said Shinoda. “We thus developed an acid-tolerant GFP, calling it Gamillus.”
Gamillus exhibits superior acid tolerance (pKa=3.4) and is nearly twice as bright compared with pre-existing GFPs. The fluorescence spectrum is constant between pH4.5 and 9.0, which falls between the intracellular range in most cell types.
X-ray crystallography, a technique used for determining the atomic and molecular structure of proteins, combined with point mutagenesis, suggested that the acid tolerance of Gamillus is attributed to stabilization of deprotonation in its chemical structure.
“The feasibility of Gamillus as a molecular tag was demonstrated by its correct localization pattern in a variety of cellular structures, including ones that are difficult to target,” said corresponding author Professor Takeharu Nagai of Osaka University. “We believe Gamillus can be a powerful molecular tool for investigating unknown biological phenomena involving acidic organelles, such as autophagy.”
The article can be found at: Shinoda et al. (2017) Acid-Tolerant Monomeric GFP from Olindias formosa.
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Source: Osaka University.
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