When it comes to protein folding, cells have a few tricks up their sleeve, report researchers from the University of Illinois at Urbana-Champaign. Using laser-based techniques, they have shown for the first time that cells strongly modulate the speed and stability of protein folding. “This provides an entirely new mechanism by which cells can control what their proteins do,” said Martin Gruebele, professor of chemistry, physics and biophysics. “In addition to folding reactions, other reactions are likely also to be modulated in the cell.” Gruebele said reactions affected by the cellular environment might be those related to Alzheimer’s, Huntington’s or mad cow diseases, all of which are characterized by protein misfolding and plaques. Other reactions that could potentially be affected because they take place inside a cell are those related to the complex protection mechanisms that arise during a fever. The cube shown here represents a magnified pixel imaged from the cell, with folded (right-pointing arrow) and unfolded (left-pointing arrow) proteins in equilibrium. The yellow structures are red acceptor and the green are donor fluorescent labels, used to measure distances and the protein state. Courtesy of Martin Gruebele, University of Illinois, Urbana. The discovery was made possible because Gruebele and his colleagues successfully combined laser-driven temperature-jump relaxation with fluorescence microscopy. Although both techniques are individually well developed, merging them presented challenges. One was how to spike the temperature up or down rapidly and then keep it constant afterward on a large microscope stage with multiple objectives, slides and other heat-conducting equipment. The solution to this and other engineering problems, as outlined in a paper published in the April 2010 issue of Nature Methods, involved multiple lasers operating at different wavelengths. For temperature jumping, the researchers used a laser emitting at 2.2 μm, employing two types of pulses. For one, they shaped the laser pulse into a spike followed by a plateau, which caused a rapid temperature rise. For the other, they preheated the cell and then dropped the laser power to zero, achieving a downward temperature jump that took less than 50 ms. They monitored the temperature profile by exciting an acceptor dye with a green diode laser, calibrating the observed intensity to an absolute temperature scale. They could do this on a pixel-by-pixel basis for the captured image. When studying protein-folding dynamics, they used a blue LED or an argon-ion laser to excite a donor attached – such as the acceptor – to the biomolecule being studied. Thanks to Förster resonance energy transfer, the resulting emission provided a molecular ruler to measure the distance between donor and acceptor. With optics, they captured the fluorescence, split it into red and green channels and imaged it using a CCD camera, recording a frame every 16.7 ms. This was the limit for the time-resolution of the equipment. A faster camera could cut that into the microsecond range, the researchers asserted. In a series of experiments, the investigators looked at protein folding of the phosphoglycerate kinase construct in two human cell lines at various temperatures. They did this in vitro, or in the equivalent of a test tube, and in a live cell. The ability to do these studies in a live cell is new and led to some interesting results, Gruebele said. “We showed that the protein is more stable in the living cell than it is in the test tube, and that the protein folds at a different rate in the living cell than in vitro.” The reason for the differences has to do with the crowded nature of the cell, he explained. In a test tube, a protein can fold without constraint. A cell, in contrast, is full of structures that act as channels and barriers that affect the rate of folding or keep it from happening altogether. Using the technique, clinical researchers might be able to get useful information about misfolding diseases, heat-shock responses or other medically interesting cellular behaviors. As for the Urbana group, they are applying the technique with a final outcome in mind, Gruebele said. “Our ultimate goal is a better understanding of how temperature affects the behavior of cells and organisms, at the cell and chemical level.”