A new method uses fluorescence to detect potentially disease-causing forms of proteins as they misfold and unravel due to stress or mutations. Researchers from Pennsylvania State University (Penn State) and the University of Washington re-engineered a fluorescent compound and developed a method by which two different proteins can fluoresce at the same time as they misfold and aggregate inside a living cell, highlighting forms that could play a role in several neurodegenerative diseases, including Alzheimer’s and Parkinson’s. The new AggTag method allows researchers to see the previously undetectable but potentially disease-causing intermediate forms of proteins as they misfold. The method uses fluorescence to simultaneously detect two different proteins (red, green) within the cell (blue). Courtesy of the Zhang Lab, Penn State. “Protein aggregation is a multistep process, and it is believed that the intermediate form, which previous imaging techniques could not detect, is responsible for a number of diseases, including Alzheimer’s, Parkinson’s, type 2 diabetes, and cystic fibrosis,” said professor Xin Zhang. “We developed the Aggregation Tag method — AggTag — to see these previously undetectable intermediates — soluble oligomers — as well the final aggregates in live cells.” The AggTag method uses “turn-on fluorescence,” so the compound only lights up when misfolding starts to occur. “When the fluorescent compound has plenty of space to move, it rotates freely and remains turned off, as in the presence of a properly folded protein,” said professor Yu Liu. “But when the protein starts to misfold and aggregate, the compound’s movement becomes restricted and it begins to light up.” To allow for the distinction between forms, the research team re-engineered the color-causing core of the green fluorescent protein (GFP), which is commonly used in imaging studies because it fluoresces when exposed to certain wavelengths of light. The re-engineered compound binds to a tag, which in turn fuses to a protein targeted for imaging. “Diffuse fluorescence indicates that intermediate oligomers are present, while small points of brighter fluorescence indicate that the denser insoluble aggregates are present,” Liu said. The research team used two different kinds of commercially available tags, Halo-tag and SNAP-tag, which when used with AggTag can induce red or green fluorescence, respectively. Because Halo-tags and SNAP-tags do not interact with each other, they can be used to simultaneously image two different proteins with the two colors. The independent fluorescence of Halo-tags and SNAP-tags allows for simultaneous visualization of two different pathogenic protein aggregates in the same cells. The team also engineered the tags so that the green and red colors can be reversed, giving researchers options for future imaging. Unlike other methods, the AggTag method is capable of detecting previously invisible misfolded soluble proteins. The team believes that it is the first application of fluorescent protein chromophores to detect protein conformational collapse in live cells. “We plan to continue developing this method so that we can signal the transition of oligomers into insoluble aggregates using a color change,” Zhang said. “This method provides a new toolbox to study protein aggregation, which is currently a highly studied topic among scientists. Hopefully this will allow us to better understand the entire process of protein aggregation and the role of each of these forms in the progression of neurodegenerative and other diseases.” The research was published in the Journal of the American Chemical Society (https://pubs.acs.org/doi/abs/10.1021/jacs.8b02176) and in ChemBioChem (https://doi.org/10.1002/cbic.201800782).