Scientists have developed a novel way to determine the fraction of a protein that is folded into its active form in a cell (1). The method involves attaching a fluorogenic tag to a small molecule that binds only the folded protein of interest and paves the way for future studies of protein folding dynamics as well as how misfolded or unfolded proteins contribute to human disease, said Jeffery Kelly of The Scripps Institute, senior author of the new paper.
“Everybody believed it would never work to have a small molecule bind a protein to measure folding because as soon as it binds, it stabilizes the protein and shifts the folding equilibrium” Kelly said.
So rather than add small molecules to actively metabolizing cells, Kelly’s team developed a protocol that essentially “freezes” protein conformations at any given time. Cells are lysed and then rapidly depleted of ATP so that chaperones can’t convert proteins between folding states. Probes are then added, fluorescing when bound to the folded protein they target. According to Kelly, any small molecule known to bind a protein can be used in the probe.
Kelly and his colleagues tested their method on two proteins—the designed enzyme retroaldolase and the human non-enzyme transthyretin, which is implicated in amyloid diseases. In both cases, the researchers were able to quantify the amount of folded protein and then compare that level to the total amount of protein, as detected by Western blotting, to determine the fraction of protein in a particular folded state. These experiments revealed that the amount of folded and active protein was lower than the total soluble fraction.
“There are hundreds of papers in the literature where cell biologists have equated soluble to folded when it comes to proteins,” Kelly said. “That’s generally an okay assumption, but we’ve shown that it’s not always the case.”
Kelly’s team is already using the new approach to screen libraries for molecules that affect protein folding. “I think many labs will pick this up now,” he said. “After you have a folding probe for your protein of interest, there are a lot of things you can look at.”
Liu Y., Tan Y.L., Zhang X., Bhabha G., et al. (2014) Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts. PNAS Early Edition February 26, 2014.