Is it folded properly? Studying protein folding with fluorescence detection

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  • Published: Jul 18, 2011
  • Author: Jon Evans
  • Channels: Detectors
thumbnail image: Is it folded properly? Studying protein folding with fluorescence detection

Reflecting on folding

By combining a spectrograph, a lamp-based fluorescence detector and capillary electrophoresis (CE), a team of Dutch pharmaceutical scientists are able to study protein folding in two complementary but entirely different ways. As well as revealing more information about how proteins fold and unfold, this technique could help pharmaceutical companies monitor the quality of their therapeutic proteins and offer a new way to study the many diseases caused by misfolded proteins, such as Alzheimer's Disease.

This current development builds on earlier work by Govert Somsen and his colleagues at Utrecht University, in which they used the same lamp-based fluorescence detector to identify whole proteins separated by CE (see Do you want the good news or the bad news?). This detector employs a xenon-mercury lamp and a fibre optic cable to focus a beam of light of a specific wavelength on a special detection cell at the end of a separation capillary. The interior surface of this detection cell is highly reflective, directing any fluorescence generated by analytes in the cell to a photodetector on the far side of the cell.


Red shift

In 2009, Somsen and his colleagues showed that this detector could detect proteins containing naturally-fluorescent amino acids such as tryptophan with a similar sensitivity to more expensive laser-induced fluorescence detectors. They have since gone on to show that this detector can do much more than simply detect proteins. Replacing the photodetector with a spectrograph produces a detector than can also monitor the precise wavelengths of light generated by the proteins, opening up the possibility of using it to study protein folding.

The reason for this is that when a protein unfolds it exposes amino acids that were previously hidden, including naturally-fluorescent amino acids, to the liquid solution they're in, altering the wavelength of light emitted by these amino acids. This produces what is known as a red-shift effect, in which the light emitted by the protein is shifted towards longer, redder wavelengths, and has been demonstrated by numerous studies. Somsen and his team are able to use their detector to monitor this red-shift effect.

When they separated a mixture of different proteins, including bovine serum albumin (BSA) and carbonic anhydrase II, in a buffer containing urea, which causes proteins to unfold, they found that they could detect characteristic red-shifts for all the proteins. But that's not all, because the spectrograph can also produce a conventional electropherogram based on the generated fluorescence, with the peak heights directly reflecting the protein concentration. When they looked at these electropherograms, Somsen and his team quickly noticed the each protein migrated slower when unfolded than when folded.


Arms widespread

Now, this finding shouldn't come as too great a surprise. An unfolded protein possesses the same charge as a properly folded version but is more spread out, so it's naturally going to travel more slowly through a liquid. In the same way that a swimmer will glide faster through the water with his arms by his side than with them spread out. But what this means is that this detector provides two completely different ways to study protein folding: the red-shift effect and the difference in migration time.

Furthermore, although these two properties are often linked, such that those proteins with the greatest red shift also show the greatest increase in migration time, they're not always. For example, BSA has a large red shift but only a relatively small increase in migration time, because it doesn't spread out too much when unfolded. So comparing these two properties can reveal quite a bit about how different proteins unfold and this is what Somsen and his colleagues are now going on to investigate.

Unfortunately, other research groups may have difficulty in following them, because as revealed in Do you want the good news or the bad news? the lamp-based detector employed by Somsen no longer appears to be commercially available.



The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

Is it folded properly?: studying protein folding with fluorescence detection

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