FRETting about folding

Nature is the consummate magician. There are things that human intelligence and its servant computers fail to do despite the mightiest struggles; nature does them with an insouciant shrug. For a biologist, the most maddening example of this is the folding of proteins.

A protein is a linear molecule, hundreds or thousands of atoms long. Every third atom in this chain has a chemical decoration; there are twenty different types of decorations, some acidic, some basic, some neutral, some positively charged, or negatively, or relatively large or small. Depending on the linear arrangement of these decorations, the protein can coil up like an old-fashioned telephone cord, or fold itself in pleats, or sort of randomly squiggle about, in any combination of different patterns in three-dimensional space. Here’s an example, the botox protein I wrote about earlier.

If this protein were to fold up in the wrong shape, it wouldn’t work at all. So how does a protein always fold up into the right shape?

Now, we know that the decision about how to bits of the protein line up next to each other is in some way dependent upon the order of decorations on the atoms in the protein chain. Some decorations like to be next to each other, while others shun each other’s company. In a really simplified image, you can imagine a protein as being like a whip with decorated with a plus and a minus static charge, a north and a south pole magnet, a bit of fuzz and a bit of claw Velcro, a “male” and a “female” Lego block, a similar pair of Duplo blocks, and an electrical plug and a socket. If you were to randomly shake that whip around, you could predict that you would always end up at the same end state: plus with minus, north with south, and so on. This arrangement is the most stable state—the lowest energy state.

Proteins (in theory) behave similarly, only the “whip” is shaken by the random jiggling of thermal motion—and, usually, a specific protein with a specific arrangement of decorations will always end up in the same three-dimensional shape. And, as a testament to human ingenuity and the power of computers, we can actually predict the most stable, lowest-energy state of short proteins with relatively simple arrangements of decorations.

We run into problems, though, when we try to predict the three-dimensional structure of more everyday proteins—which have hundreds of decorations. The most sophisticated computers get bogged down with all the possible permutations, and we have a mixed record at best for understanding how these things fold up. And while we crack our skulls about the problem, nature casually takes proteins and effortlessly folds them into the right shape, over and over again. It keeps a biologist humble.

We don’t even really understand the kinetics of the process—some proteins fold up into their finished shape in milliseconds, while others that are not much longer take thousands of times longer to fold up. What takes longer? Do the slower proteins have more possibilities to try out before they settle on the best shape? Are they just not as flexible? A neat technical tour-de-force gives us at least a little clue towards this last problem. A group of researchers at the National Institutes of Health (I approve of this use of my taxes) found an interesting similarity between the behavior of “fast-folding” and “slow-folding” proteins.

So, consider these two proteins.

This one, nicknamed WW, folds into this shape rapidly.

This one, named GB1, folds into shape 10,000 times more slowly.

What does that mean, what I just wrote? Those numbers are based on taking a huge number of unfolded protein molecules of WW or GB1, putting them in solution, and measuring how long it takes for half of them to assume their folded shape. So, in this case, “how fast something folds” is descripting of a large population, but doesn’t tell us much about how an individual molecule behaves. How long does it take a single individual protein to transition from unfolded to properly-folded?

A morbid analogy would be the half life of a human population: if you looked at all the people born in 1903, you could calculate a half-life, or how long it takes for half of that group of people to die. This tells you a lot about how long an average person lives, which is many years. It tells you nothing about how long it takes to transition from alive to not-alive, which is usually a rapid transition.

What the NIH researchers did was to modify these proteins so that they could examine them, and distinguish more precisely how long it took an individual to change from unfolded to properly folded. To do this, they attached specific dye molecules to either end of the unfolded protein. These dye molecules have a really cool property: if you zap one with the right amount of energy in the form of purple light, it will actually dump that energy onto the other dye molecule, which will fluoresce, shining with red light. They will only do this, though, if the dye molecules are really close together, and in this setting, they are only close together if the protein is properly folded. This process is called Förster Resonance Energy Transfer, or FRET. To go back to the image of a decorated whip, this is like attaching dye markers to either end of the whip. If the whip is unfolded, when you shone purple light on it, it wouldn't fluoresce. Energy couldn't get from one end of the whip to the other:

If it were partially folded, it would fluoresce weakly, because it would be hard for energy to get from one dye molecule to the other.

If it were fully folded, and you shone purple light on it, it would be easy for energy to get from one dye molecule to the other, so it would fluoresce brightly.

So, you could measure how fast it takes the whip to get folded by measuring how rapidly red fluorescence increases. So, measuring how long it takes an individual protein to change from unfolded to properly folded was a matter of measuring how rapidly FRET efficiency increased.

The data from these experiments are not all that fun to look at, involving a fair amount of

But the bottom line was that for both fast- and slow-folding proteins, the transition from no structure at all to completely folded structure was about the same, in the range of a hundredth of a millisecond. The "slow-folding" proteins seem to dawdle and delay and do everything they can to put off folding, but once they decide to fold, they fold just as rapidly as the "fast-folding" ones. It's kind of like the situation mentioned earlier with the population of humans born in 1903; some may live a long time, others die in infancy, but the transition between alive and dead always takes the same, brief amount of time.

So, we know a little more about the process of protein folding now. If we are trying to understand why two proteins fold up at rates that differ ten thousand-fold, we at least know where not to look for answers. However, we still don’t really know what the answer is--what the slow protein is doing when it's not folding up. Nature, like a good magician, is still reminding that we are in the dark.

Hoi Sung Chung, Kevin McHale, John M. Louis, and William A. Eaton (2012). Single-Molecule Fluorescence Experiments Determine Protein Folding Transition Path Times. Science 335: 981- 984.

The Wikipedia web page on FRET is not bad. The above is obviously a gross simplification.