Scientists have long puzzled over the following conundrum: If the activity of a protein solution drops by half, have half the molecules completely lost activity, or have all the proteins lost half their activity? The answer, most likely, is that the solution comprises a mixture of proteins existing in many intermediate states between fully active and inactive, but proving this would require the study of single molecules, which, until recently, was a daunting task.

Advances have been made however, most notably in the measurement of single molecules of luciferases, enzymes aptly suited to such measurements because they emit photons that are relatively easy to detect. In today's Science, U.S. and German researchers exploit the sensitivity of photon-detecting devices to take the study of single molecules one step further, measuring not protein activity, but folding kinetics.

Led by William Eaton from the NIDDK, Bethesda, Maryland, the researchers capitalized on a phenomenon called Forster Resonance Energy Transfer, or FRET (more usually called fluorescence resonance energy transfer). FRET is usually used to measure the proximity of fluorescent dyes attached to independent, though interacting proteins. Dye on one protein, excited by light of an appropriate wavelength, emits a photon that excites a second dye on another protein. This second dye then emits light of a slightly lower wavelength. Because the probability of the second emission is a function of the distance between the two dyes, FRET allows the proximity of the proteins to be easily determined.

First author Everett Lipman and colleagues used a slight variation on this theme to measure folding of the small, b-barrel, cold-shock protein from the bacterium Thermotoga maritima-they added dyes to different ends of the same molecule. When the protein is in the native state, coiled like a spring, the intramolecular FRET should be intense, but if denatured and stretched out, the FRET should weaken.

To measure single molecules, Lipman and colleagues used a miniature continuous flow device coupled to an exquisitely sensitive confocal microscope. By pumping dilute protein solutions across the field of the objective lens, the authors ensured that they were only measuring fluorescence emission from one molecule at a time. To denature the protein, Lipman pumped a concentrated solution of guanidinium chloride into the laminar flow device, while a buffered solution pumped in slightly downstream of that diluted the denaturant, allowing the protein to revert back to its native conformation.

To determine the kinetics of this renaturation, Lipman took FRET measurements at different distances from the mixing device and, hence, different times after mixing. The authors found that after about 100 milliseconds, the FRET efficiency jumped from 50 to 60 percent. The authors attribute this slight increase to a compaction of the unfolded molecules once the viscous denaturant has been removed. Over the next four seconds or so, the FRET efficiency increased to nearly 100 percent for most of the molecules, though statistically some molecules were still denatured by that time. The power of the technique, suggest the authors, is that "such subpopulations can be studied under conditions far from equilibrium, where they exist only briefly." It may also prove useful for those studying protein-folding disorders such as prion diseases and other neurodegenerative diseases, like Alzheimer's, where protein misfolding is thought to play a major role.—Tom Fagan

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Primary Papers

  1. . Single-molecule measurement of protein folding kinetics. Science. 2003 Aug 29;301(5637):1233-5. PubMed.