Expanding protein structure horizons

A new study reveals that the static snapshots recorded in protein crystallography may be missing the bigger picture. Investigations of a bacterial protein using cryomicroscopy shows the protein in a balloon-like mode previously hidden from sold state studies. The discovery suggests that techniques complementary to X-ray crystallography are essential if molecular biology is to gain a complete understanding of protein structure.

Steven Ludtke, assistant professor of biochemistry and molecular biology and co-director of the National Center for Macromolecular Imaging at Baylor College of Medicine and colleagues Dong-Hua Chen and Wah Chiu there and Jiu-Li Song and David Chuang at The University of Texas Southwestern Medical Center in Dallas, studied a mutant protein and came to this perhaps not so startling conclusion. The protein GroEL chaperones misfolded proteins and nudges them into their active folded state in the cell. Protein misfolding is implicated in a number of neurodegenerative diseases, such as Alzheimer's disease and the prion diseases including Creutzfeldt-Jakob disease.

The team used electron cryomicroscopy to obtain detailed two-dimensional images of individual molecules in solutions mimicking physiological conditions. They then used a computational reconstruction procedure to assemble tens of thousands of these images into a three-dimensional structure to reveal the dynamics of the protein in question.

In an environment similar to that in which they exist naturally, proteins and multiprotein assemblies may behave differently to the way static solid-state structures would suggest, Ludtke suggests. The results of his electron cryomicroscopy investigation would suggest that dynamic behaviour can indeed deviate from the accepted norm.

Ludtke and his colleagues investigated the mutant form of GroEL, GroES. They confirmed the existence of two structures seen in crystallographic results, but they also found a third - "a strange-looking structure blown up like a balloon" that had not been seen before. In terms of native GroEL, the finding may indicate the need to look at the chaperone itself more closely, the researchers say.

"The expansion was directly related to the function of the assembly. From a more global perspective, this is strong evidence that we need to study how any macromolecule behaves in a solution environment," says Ludtke. Of course, this particular enzyme may be exceptional. There is a chance that the "ballooned" conformation is not a natural one and that it could not revert to its original form in the cell. Ludtke, however, is convinced that there are transitional states that would allow the protein to refold into a more familiar structure. The ballooned state is large enough to hold a substrate of up to 86 kilodaltons, that is about 80 percent bigger than the substrate limit in the crystal structure.
So, is this the end for crystallography? "I am definitely not saying that the structures
produced by crystallography are incorrect," emphasises Ludtke, "just that they don't present a complete picture." The present technique represents a complementary approach that could broaden crystallographic horizons and provide additional insights into protein behaviour. Ludtke told SpectroscopyNOW that, "In essence in single particle imaging we are able to directly examine dynamics of large macromolecular assemblies, somewhat akin to NMR of small molecules."

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