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Tryptophan technique illuminates protein folding.

Misfolded proteins are implicated in diseases from Alzheimer's to Parkinson's, but tracking the process by which they occur remains one of biochemistry's greatest challenges. Now, a team at Universite de Montreal has shown that a technique based on the fluorescence of tryptophan might be a better tool to probe protein folding than anyone previously thought.

The transitions involved in protein folding are notoriously difficult to study as the half-folded intermediates don't usually last long enough for their unique signatures to be unambiguously detected by traditional methods such as crystallography or nuclear magnetic resonance (NMR) spectroscopy. An alternate method is based on the fluorescence of tryptophan (while several amino acids exhibit fluorescence, tryptophan's is the strongest). By measuring changes in the light emitted by excited tryptophan molecules, researchers can glean information about the local environment in a specific part of the protein.

"It has become dogma that tryptophan has to be at least partially buried in the folded structure in order to see a strong change in fluorescence between unfolded and folded states," says Stephen Michnick, a biochemist at Universite de Montreal. In a technical report published in Nature Structural and Molecular Biology Michnick and Alexis Vallee-Belisle disproved that theory. They created mutant versions of the protein ubiquitin with tryptophan substituted in sites that were exposed on the surface of the protein. Fluorescence spectroscopy showed that even on these sites, the electronic differences between folded and unfolded states was still enough to cause detectable changes in fluorescence. The team went on to create mutant versions of ubiquitin with up to 27 of its 76 amino acids replaced with tryptophan. The larger number of probes allows researchers to study many areas of the protein at once.


The technique is surprisingly simple, which Michnick says is precisely the point. "I hope this gives the protein community a license to try something that they probably wanted to try but didn't have the nerve to, because they thought it was crazy," he says. He adds that the technique could be applied not only to protein folding intermediates, but any conformational change in proteins including allosteric transitions and macromolecular assembly.
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Title Annotation:BIOCHEMISTRY
Publication:Canadian Chemical News
Date:Sep 1, 2012
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