Proteins may behave differently in fluids

Proteins and macromolecular structures may take on unexpected arrangements in natural fluid environments not seen in the static crystalline surroundings in which they are typically studied, according to a report in Structure.

Steven Ludtke, co-director of the National Center for Macromolecular Imaging at Baylor College of Medicine and colleagues observed GroEL/GroES, a well-studied bacterial chaperonin complex that ensures proper protein folding, using cryoelectron microscopy, a technique that reconstructs three-dimensional protein structures in ultracooled samples.

The researchers found two GroEL/GroES structures in solution that were not surprising based on previous X-ray crystallographic studies. However, they also found a third configuration never seen before, which Ludtke described as "a strange-looking structure blown up like a balloon."

This result suggests that snapshots of macromolecular structures taken with methods such as crystallography may not be helpful in terms of understanding how dynamic protein complexes behave in their more native fluid environments.

"From a more global perspective, this is strong evidence that we need to study how any macromolecule behaves in a solution environment," Ludtke said. "This sort of expansion has never been observed before."

Hays Rye, a biochemist at Princeton University, told The Scientist, "A lot of these macromolecular complex machines may have dynamics and degrees of freedom and plasticity that we only get a glimpse of by looking at one or two structures of them."

However, some researchers expressed caution about the new results. The chaperonin under examination typically forms a GroEL double-ringed structure capped with GroES. Ludtke's group used a mutant version of GroEL, SR1, which forms a single-ring structure that could be responsible for the complex's expandability.

The double-ring's interface constrains the structure, according to Art Horwich, a structural biologist at Yale University who works on GroEL. "[The mutant GroEL] SR1 has always been a candidate for structural ballooning like this that is not physiological," he told The Scientist. Horwich said that an important next step would be to determine if single-ringed mammalian mitochondrial chaperonins have this level of flexibility. "The question in my mind is: After the ring has undergone that kind of engorgement and distortion, can it ever go back to the original state?"

Ludtke argues that the ring likely does return to its original state, because the cryo EM study shows intermediates of the "normal" to ballooned forms of GroEL, suggestive of transient states. Further, "we don't see any non-expanded structures that have the substrate inside," he said.

Also at stake is the question of chaperonin's upper limit of substrate size. So far, experiments put that limit at 60 kDa. However, Studtke's group found that GroEL was able to handle a heterodimer of 86 kDa. Studtke only observed the ballooned structures in the presence of the large heterodimer.

"It opens up a possibility that would be extremely exciting, that GroEL can morph to accommodate larger structures," said Lila Gierasch, a biophysicist who works on GroEL at the University of Massachusetts Amherst. Beyond the single-ring problem, Gierasch said another worry is that the researchers imposed averaging to ensure their complex resembled the expected seven-sided structure. Once these two technical hurdles are overcome, "it could change a paradigm about which populations of newly synthesized proteins are substrates of GroEL," she said.

Already, GroEL's substrate limits have begun to expand. Horwich's lab recently showed in a report in PNAS that substrates larger than 60 kDa can get folding assistance from GroEL and that, in fact, GroEL may assist the majority of newly synthesized polypeptides, regardless of size. However, Horwich's model does not depend on proteins entering into GroEL's channel, in contrast to Ludtke's hypothesis.

The middle-ground may be that the ballooned single-ring structures simply exaggerate the dynamics found in the double-ring, according to biochemist Zhaohui Xu at University of Michigan Medical School, who solved the GroEL-GroES-(ADP)7 chaperonin complex in the late 90s. "Without knowing what's happening in the double-ring, it's tough to determine how biologically relevant this is," Xu said.

Trevor Stokes
mail@the-scientist.com

Links within this article:

Chen et al., "An Expanded Conformation of Single-Ring GroEL-GroES Complex Encapsulates an 86 kDa Substrate," Structure (2006), doi:10.1016/j.str.2006.09.010
http://www.structure.org

Steven Ludtke
http://ncmi.bcm.tmc.edu/homes/stevel

National Center for Macromolecular Imaging at Baylor College of Medicine http://ncmi.bcm.tmc.edu/ncmi/

Hays Rye
http://www.molbio2.princeton.edu/index.php?option=com_content&task=view&id=228

N. Sankaran, "Hot Paper: Biochemistry/ Structural Biology," The Scientist, March 4, 1996
http://www.the-scientist.com/article/display/16920/

Art Horwich
http://info.med.yale.edu/genetics/horwich

Lila Gierasch
'http://www.biochem.umass.edu/gierasch/

Champan E, et al., "Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL," PNAS, Oct 24; 103(43): 15800-5. Epub 2006 Oct 16
http://www.pnas.org/cgi/content/full/103/43/15800

Zhaohui Xu
http://www.biochem.med.umich.edu/biochem/research/profiles/xu.html