Year: 2024
Type: editorial
Abstract: My first exposure to visibly fluorescent proteins (FPs) was near the end of my time as a faculty member at the University of California, Berkeley.Prof. Alexander Glazer, a friend and colleague there, was the world's expert on phycobiliproteins, the brilliantly colored and intensely fluorescent proteins that serve as light-harvesting antennae for the photosynthetic apparatus of blue-green algae or cyanobacteria.One day, probably around 1987-88, Glazer told me that his lab had cloned the gene for one of the phycobiliproteins.Furthermore, he said, the apoprotein produced from this gene became fluorescent when mixed with its chromophore, a small molecule cofactor that could be extracted from dried cyanobacteria under conditions that cleaved its bond to the phycobiliprotein.I remember becoming very excited about the prospect that an arbitrary protein could be fluorescently tagged in situ by genetically fusing it to the phycobiliprotein, then administering the chromophore, which I hoped would be able to cross membranes and get inside cells.Unfortunately, Glazer's lab then found out that the spontaneous reaction between the apoprotein and the chromophore produced the "wrong" product, whose fluorescence was red-shifted and five-fold lower than that of the native phycobiliprotein [1][2][3] .An enzyme from the cyanobacteria was required to insert the chromophore correctly into the apoprotein.This enzyme was a heterodimer of two gene products, so at least three cyanobacterial genes would have to be introduced into any other organism, not counting any gene products needed to synthesize the chromophore 4 .Meanwhile fluorescence imaging of the second messenger cAMP (cyclic adenosine 3',5'-monophosphate) had become one of my main research goals by 1988.I reasoned that the best way to create a fluorescent sensor to detect cAMP with the necessary affinity and selectivity inside cells would be to hijack a natural cAMP-binding protein.After much consideration of the various candidates known at the time, I chose cAMP-dependent protein kinase, now more commonly abbreviated PKA.PKA contains two types of
Source: PLoS Computational Biology
Institutions: University of Virginia, Johns Hopkins University, Yale University
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,926
OpenAlex ID: W4402898775