Scientists at University of Illinois at Urbana-Champaign are now able to monitor the folding of proteins within living cells, giving scientists a new tool to monitor protein behavior. Because a higher level of complication is involved within a cell than can be reproduced in a test tube, this technique may give a truer understanding of what proteins are up to.
To study the biomolecular dynamics inside of a single living cell, Gruebele [Martin Gruebele, professor of chemistry at University of Illinois] and his team pioneered a hybrid method they’ve dubbed “Fast Relaxation Imaging,” a technique that combines fluorescence microscopy and fast temperature jumps.
To achieve both a fast upward and downward temperature jump, programmed laser pulses are used to pre-heat, spike, plateau, cool and then finally stabilize the temperature in the cell and its aqueous medium at the final value. An inverted fluorescence microscope is used to observe and record what happens inside the cell, all of which takes place in the span of a few milliseconds.
The cells are usually heated to between 96 and 100 degrees Fahrenheit.
“It’s like we give them a little bit of a fever,” Gruebele said.
Gruebele says that although temperature jumps have been used for some time to study the kinetics of chemical reactions in vitro, that method is limited by what he calls “homogenous kinetics,” or an inability to see the dynamics in different areas of the cell.
“We haven’t really been able to study dynamics, to see if a chemical reaction like protein folding varies inside of a living cell,” he said. “With temperature jumps and pressure jumps, you can do those experiments very quickly, but you don’t get any imagery that lets you see if proteins fold faster in one region and slower in another,” Gruebele said.
On the other hand, fluorescence microscopy allows researchers to see inside of cells, but it precludes them from studying cell dynamics and kinetics.
“With fluorescence microscopy, we’re able to take images of cells and see inside them, but we can’t observe how anything rapidly changes or adapts with time, so you can’t look at any but the slowest dynamics. This experiment puts those two aspects together,” he said.
Since biomolecular dynamics are predominantly studied in vitro, with the results extrapolated to explain how the same processes would function in a living cell, Gruebele says the new technique has yielded some interesting data that could change standard thinking in the field.
Abstract in Nature Methods: Protein folding stability and dynamics imaged in a living cell
Press release: New technique allows study of protein folding, dynamics in living cells …