Check the cover of any biochemistry textbook or peer-reviewed journal, odds are you'll see protein structures, cartoon representations that reduce the molecules’ tens of thousands of individual atoms into simplified sculptures resplendent in coils and ribbons of red, blue, green and yellow.
Generally solved using X-ray crystal-lography, nuclear magnetic resonance spectroscopy, or cryoelectron microscopy, such simplified structures help researchers conceptualize macromolecular protein complexes, visualize the impact of mutations, and even design targeted drug therapies. There's just one problem with these structure s: Proteins are not as rigid as their structural depictions imply. Instead, they breathe and flex, folding and unfolding in response to changing cellular conditions, post-translational modifications, and simple thermodynamics.
“People have this idea that when a protein folds in the cell, it folds once and that's the end of it. But that's not true,” explains Martin Gruebele, who studies protein folding at the University of Illinois at Urbana- Champaign. “Proteins fold and unfold and fold and unfold in the cell…. They continuously go back and forth.”
While the pretty pictures published on book covers and journals are indeed accurate, they only tell part of the story. These images don't represent every possible form of the molecule, or perhaps even the most biologically interesting ones. Rather these are the most stable or crystallizable states, what North Carolina State University physicist Keith Weninger calls “landmarks in a conformational landscape.”
And that's just in vitro; what a protein looks like in vivo may differ even more. “Living cells are amazing things,” Weninger says. “They maintain non-equilibrium conditions; the system keeps gradients that shouldn't exist, and very non-equilibrium flows, and those are hard to reproduce outside of a cell. Those conditions can affect biology, which is why people want to develop high-resolution methods to look at protein structure in cells.”
Until recently researchers had no way to capture such data. But today, using a crop of new fluorescence and NMR techniques, researchers are finally catching glimpses of protein structures and even folding events in cells rather than in a test tube.
Single-Molecule FretTo watch proteins go through their thermodynamic acrobatic routines in vivo, both Gruebele and Weninger have turned to fluorescence resonance energy transfer (FRET, also called Förster resonance energy transfer). FRET uses the efficiency of non-radiative energy transfer between two fluorophores as a measure of the distance between them.
Imagine you position fluorophores at either end of a peptide that is capable of folding into a hairpin. One fluorophore absorbs blue light and emits green while the other absorbs green light and emits red. In an unfolded, extended conformation, irradiation of your peptide with blue light will produce a green fluorescent signal, as expected. But, if the molecule folds properly, and the two fluorophores are brought together in close proximity, then excitation with blue laser light will yield red fluorescence, as the green fluorophore passes its energy to the second dye.
The efficiency with which FRET occurs is a sensitive measure of the distance between the two points, leading some to call the technique a “molecular ruler.” As with physical rulers, the resulting measurements can be used to pin down molecular structures. For instance, in one recent study Weninger and his team used 34 separate FRET distance measurements to “constrain” a structure of the synaptotagmin 1-SNARE complex, a multicomponent assembly that could not be solved by other methods. “To our knowledge, this is the first experimentally derived model of a synaptotagmin–SNARE complex, which has resisted crystallization and NMR analysis,” the authors wrote. (1)
But that work was done in vitro; studying structure in vivo, Weninger says, is more challenging. “It's noisier, less controlled, so it's harder to interpret the data, make measurements, get good signal-to-noise ratios, and so on.” Nevertheless, Weninger has begun migrating his studies into cells, using FRET to monitor the folding of a protein called SNAP-25.
SNAP-25 is a SNARE protein, a family of polypeptides implicated in membrane fusion. As a monomer, SNAP-25 remains relatively unfolded. But when the protein enters a membrane fusion complex with other SNARE proteins, SNAP-25, well, snaps into a tightly folded helical bundle conformation. It's a molecular event akin to an unfolded tangle of yarn suddenly ordered itself into a skein.
