Marking Folded Proteins for Basic Alzheimer’s Studies

The new technique should allow labeling of correctly folded proteins in a living cell or similar natural environment to study the origins of protein-misfolding diseases such as cystic fibrosis, Alzheimer’s and Parkinson’s.

Findings of Gierasch, a distinguished professor of biochemistry and molecular biology, and Beena Krishnan, a postdoctoral fellow, appear today in the journal, Chemistry and Biology.

Cells, once thought to be simple watery bags, are now understood to be more like a thick porridge of protein chains, nucleic acids, membranes and other components. This makes it extremely difficult to observe the delicate protein-folding process, according to Gierasch, who says it’s like trying to watch knot-tying in a microscopic bowl of spaghetti. By discovering how to mark certain protein segments with naturally fitting fluorescent inserts, she and Krishnan have created what they call a “structure sensor.” It lights up only when the protein is perfectly folded, allowing it to be seen against the background of cellular complexity.

Proteins carry out thousands of normal cell operations by folding themselves into different three-dimensional, origami-like shapes matched to a single job as a key fits a lock. It’s believed that misfolding occurs regularly and when a cell detects mistakes, it just recycles protein parts and uses them again. However, if a certain misfolded key somehow gets past the cell’s “quality control,” Gierasch notes, proteins may aggregate or clump, with devastating results that include neurodegenerative diseases and other pathologies.

Fluorescent marking isn’t a new technique, but Gierasch and Krishnan applied it in a new way, snipping a segment of naturally occurring protein and replacing it with what they dub a “cross-strand tetra-Cys motif.” When the two parts of the motif settle near each other when properly folded, the dye binds and gives off light. Interestingly, the FlAsH dye Krishnan and Gierasch used was developed by Roger Tsien, winner of the recently announced 2008 Nobel Prize in chemistry.

Medical researchers around the world who use the new method will be able to set up a series of protein-folding experiments. By varying factors in each experiment and using the “structure sensor” to check results, they’ll piece together how protein misfolding leads to disease.

“We took on the challenge in 2004 to look for a way to see a protein inside a living cell and watch as it folds without interfering with the natural process,” Gierasch notes. Their laboratory is one of only a handful around the country working on the problem. “Clinical researchers know that you can get sick from mistakes in folding, but we don’t know how, because until now we couldn’t begin to watch the correct cascade or pinpoint mistakes in it, in order to understand the illness.” In 2006, Gierasch received a National Institutes of Health Director’s Pioneer Award of $2.5 million for her research on protein folding in the cell.