Three University of Massachusetts-Amherst (UMass Amherst) scientists will study folding and misfolding of secretory proteins in the cell’s protein factory, the endoplasmic reticulum, where misfolding can lead to diseases such as cystic fibrosis and liver cirrhosis. They have also received a four-year, $1.2 million EUREKA grant from the U.S. National Institutes of Health (NIH) to complete this work.
Anne Gershenson and her biochemistry and molecular biology colleagues Daniel Hebert and Lila Gierasch begin pilot studies this month using fluorescence resonance energy transfer (FRET) to observe how individual secretory proteins fold; not only in real cells, but in real time. For this work, they will also use a powerful, new super-resolution fluorescence microscope built by UMass Amherst physicist Jennifer Ross and her students. It provides a clear view of individual molecules with far more precision than was possible using traditional light microscopy.
In using FRET, two differently colored fluorescent dyes that are sensitive to each other, a donor and an acceptor, are introduced into the protein chain as it is made. When these are farther apart in a not-yet-folded protein, the donor emits greener fluorescence. As the protein folds and the two dyes get closer together on the three-dimensional structure, their proximity results in emission of a redder fluorescence. Spectroscopically analyzing the fluorescence allows investigators to precisely track donor-to-acceptor distance changes.
|Fluorescence microscope image shows isolated endoplasmic reticula. The cartoon illustrates the proposed experiments by Anne Gershenson and colleagues where FRET (green and red dyes) will be used to monitor the protein structure (blue chain) while it is synthesized and folds inside a single endoplasmic reticulum. (Image courtesy Anne Gershenon)|
Further, by introducing the FRET donor and acceptor molecules at slightly different parts of the protein chains in each of hundreds of experiments, Gershenson and colleagues will be able to map protein changes associated with folding in situ, in real time.
“Once we get the system operating with good fluorescence and we become adept at incorporating the FRET dyes, our method should prove useful to other researchers for studying a wide variety of other proteins which are involved in many other disease processes,” she says. “That’s the big promise of this approach, and the challenge.”
Over the three years of the program, NIH has awarded 56 grants totaling $67.4 million to support these highly innovative research projects, which promise big scientific payoffs. Awards announced total $25.2 million to 21 institutions.
Posted by Lee Mather