Biology Professor Judith Frydman and graduate student Nicholai Douglas, who was first author on the paper published in Cell. (Credit: L.A. Cicero), March 16, 2011When vital proteins in our bodies are misfolded, debilitating diseases can result. If researchers could see the folding happen, they might be able to design treatments for some of these diseases or even keep them from occurring. But many of our most critical proteins are folded, hidden from sight, inside tiny molecular chambers. Now researchers at Stanford have gotten the first-ever peek inside one of these protein-folding chambers as the folding happened, and the folding mechanism they saw surprised them.


Misfold an origami swan and the worst that happens is you wind up with an ugly paper duckling. Misfold one of the vital proteins in your body — each of which must be folded in a particular way to perform its function — and the result can be a debilitating neurodegenerative disease such as Alzheimer’s or Huntington’s.

There are no cures for such brain-wasting diseases, but now Stanford researchers have taken an important step that may one day aid in developing therapies for them. They have literally popped the lid off one of the microscopic chambers in which many of life’s most crucial proteins are folded, witnessing a surprising mechanism as the heretofore hidden folding process happened before their eyes.


Virtually all proteins need to be folded, whether in primitive organisms such as bacteria or multicellular creatures such as humans. Many are guided through the process by molecules called chaperones, of which a specialized subset — chaperonins — folds many of the most complex proteins.


Folding in bacteria has been studied in detail, but Judith Frydman, a professor of biology who led the Stanford research, said this is the first time anyone has seen the folding process performed in higher organisms.


“The mechanism of folding we saw in the chaperonin is very different from what we expected and from what has been seen in bacteria,” Frydman said. “It was really surprising, and we are still amazed that it worked. This chaperonin appears to provide a unique chemical environment.”


Chaperonins are shaped like a barrel, with two ring-shaped chambers arranged one atop the other. At the open end of each ring is a lid that opens and closes in a spiraling fashion, like the aperture of a camera, something Frydman’s team discovered in 2008 while studying the chaperonin called TRiC. Since then, they’ve been working to solve the puzzle of how a protein gets folded once the chaperonin has grabbed it, pulled it into the chamber and the aperture has closed. A paper describing their findings was published earlier this year in Cell.


Frydman said there were two likely ways in which a protein, initially a linear chain of molecules (amino acids), could theoretically be folded inside the chamber.

One is by mechanical means, with the chamber holding onto the protein and physically pushing it into the right shape.


“The other one is that when the lid closes, the chaperonin lets go of the protein, but some special chemical properties in this chamber somehow make it fold,” she said. “Our evidence is that this mechanism is the correct one.”


The only way to know which mechanism was doing the work was to see inside the chamber while the folding was happening, but simply opening up the lid wouldn’t work, because the shape of the entire chamber changes in accordance with the motion of the lid. When the lid spirals open, the walls of the chamber spiral open, too, and the protein floats away.


To see what was happening, Frydman’s team devised a chemical “trick” by which they could remove the lid on the chamber, but still get the walls of the chamber to close in, as if the lid were spiraling.

When they “closed” the lidless chamber, the chaperonin simply released the protein that had been destined to be folded. Like a long balloon that slipped from a child’s grip before it could be folded into a giraffe, the protein simply drifted off.

The challenge then became figuring out how the protein was getting released.

“One of the reasons why the mechanical model of pushing the protein into shape without letting go had been proposed was because there was no obvious way for this chaperonin to let go of the protein,” Frydman said.


When a protein gets grabbed for folding by TRiC, it is held by eight binding sites along the walls of the chamber. Between each binding site is a tiny loop. Frydman’s team suspected that during the closing process, the loops might move to somehow “shave off” the protein and release it into the folding chamber. One of her students made mutations in the loop. When the researchers did experiments in which TRiC chaperonins equipped with mutated loops were closed, the protein stayed put. It also failed to fold.


“That suggests that the way this chaperonin folds its proteins is by releasing them in a closed chamber that has very special chemical properties,” Frydman said.


“This mechanism of release is completely different from what has been seen in any other chaperone. That was very, very surprising.”


The experimental work described in the Cell paper was done using a simpler version of TRiC, from a single-celled organism, than would be found in multi-cellular organisms, Frydman said, because the simpler version is much easier to manipulate.


“Now we are interested in going back to the eukaryotic [multi-cellular] complex, where every binding site in the folding chamber is different and every release loop is different,” Frydman said. “I think this really opens up a lot of interesting avenues to explore how this works in higher organisms. Since TRiC helps fold many disease-linked proteins, and is central to protect cells from misfolding diseases such as Huntington’s disease, this work could have many therapeutic applications.”

Story Source:

The above story is reprinted (with editorial adaptations by ScienceDaily staff) from materials provided by Stanford University. The original article was written by Louis Bergeron.

Journal Reference:

1.                       Nicholai R. Douglas, Stefanie Reissmann, Junjie Zhang, Bo Chen, Joanita Jakana, Ramya Kumar, Wah Chiu, Judith Frydman. Dual Action of ATP Hydrolysis Couples Lid Closure to Substrate Release into the Group II Chaperonin Chamber. Cell, 2011; 144 (2): 240 DOI: 10.1016/j.cell.2010.12.017, March16, 2011 — A new study involving the University of Colorado Boulder shows clear evidence of the continuous control of fire by Neanderthals in Europe dating back roughly 400,000 years, yet another indication that they weren’t dimwitted brutes as often portrayed.


The conclusion comes from the study of scores of ancient archaeological research sites in Europe that show convincing evidence of long-term fire control by Neanderthals, said Paola Villa, a curator at the University of Colorado Museum of Natural History. Villa co-authored a paper on the new study with Professor Wil Roebroeks of Leiden University in the Netherlands.


“Until now, many scientists have thought Neanderthals had some fires but did not have continuous use of fire,” said Villa. “We were not expecting to find a record of so many Neanderthal sites exhibiting such good evidence of the sustained use of fire over time.”


A paper on the subject was published in the March 14 issue of the Proceedings of the National Academy of Sciences.


Neanderthals are thought to have evolved in Europe roughly 400,000 to 500,000 years ago and went extinct about 30,000 years ago. Neanderthals ranged over much of Europe and stretched to Central Asia. Neanderthals were stockier than anatomically modern humans and even shared the same terrain for a time, and there is evidence that contemporary humans carry a small amount of Neanderthal DNA. Modern humans began migrating out of Africa to Europe some 40,000 years ago.


Archaeologists consider the emergence of stone tool manufacturing and the control of fire as the two hallmark events in the technological evolution of early humans. While experts agree the origins of stone tools date back at least 2.5 million years in Africa, the origin of fire control has been a prolonged and heated debate.


Villa and Roebroeks, who together speak and read six languages, have visited or worked at dozens of the Neanderthal excavation sites in Europe. They also combed libraries throughout Europe and the United States for research papers on evidence for early fire use in Europe, contacting researchers involved in the excavations when possible for additional information and insight.


As part of the study they created a database of 141 potential fireplace sites in Europe dating from 1.2 million years ago to 35,000 years ago, assigning an index of confidence to each site. Evidence for the sustained use of fire includes the presence of charcoal, heated stone artifacts, burned bones, heated sediments, hearths and rough dates obtained from heated stone artifacts. Sites with two or more of the characteristics were interpreted as solid evidence for the control of fire by the inhabitants.


The second major finding in the PNAS study — perhaps even more surprising than the first — was that Neanderthal predecessors pushed into Europe’s colder northern latitudes more than 800,000 years ago without the habitual control of fire, said Roebroecks. Archaeologists have long believed the control of fire was necessary for migrating early humans as a way to reduce their energy loss during winters when temperatures plunged below freezing and resources became more scarce.


“This confirms a suspicion we had that went against the opinions of most scientists, who believed it was impossible for humans to penetrate into cold, temperate regions without fire,” Villa said.


Recent evidence from an 800,000-year-old site in England known as Happisburgh indicates hominids — likely Homo heidelbergenis, the forerunner of Neanderthals — adapted to chilly environments in the region without fire, Roebroeks said.


The simplest explanation is that there was no habitual use of fire by early humans prior to roughly 400,000 years ago, indicating that fire was not an essential component of the behavior of the first occupants of Europe’s northern latitudes, said Roebroeks. “It is difficult to imagine these people occupying very cold climates without fire, yet this seems to be the case.”


While the oldest traces of human presence in Europe date to more than 1 million years ago, the earliest evidence of habitual Neanderthal fire use comes from the Beeches Pit site in England dating to roughly 400,000 years ago, said Villa. The site contained scattered pieces of heated flint, evidence of burned bones at high temperatures, and individual pockets of previously heated sediments.


Neanderthals, like other early humans, created and used a unique potpourri of stone tools, evidence that they were the ancient inhabitants of particular sites in Europe.


The sites catalogued by the team were dated by several methods, including electron spin resonance, paleomagnetism and thermoluminescence. Some research teams also have used microscopic studies of sediment at sites to confirm the presence of ashes. While some of the best evidence for controlled use of fire in Europe comes from caves, there are many open-air sites with solid evidence of controlled fire, they said.


According to Villa, one of the most spectacular uses of fire by Neanderthals was in the production of a sticky liquid called pitch from the bark of birch trees that was used by Neanderthals to haft, or fit wooden shafts on, stone tools. Since the only way to create pitch from the trees is to burn bark peels in the absence of air, archaeologists surmise Neanderthals dug holes in the ground, inserted birch bark peels, lit them and covered the hole tightly with stones to block incoming air.


“This means Neanderthals were not only able to use naturally occurring adhesive gums as part of their daily lives, they were actually able to manufacture their own,” Villa said. “For those who say Neanderthals did not have elevated mental capacities, I think this is good evidence to the contrary.”


Many archaeologists believe Neanderthals and other early hominids struck pieces of flint with chunks of iron pyrite to create the sparks that made fire and may well have conserved and transported fire from site to site.


Some anthropologists have proposed that Neanderthals became extinct because their cognitive abilities were inferior, including a lack of long-term planning, said Villa. But the archaeological record shows Neanderthals drove herds of big game animals into dead-end ravines and ambushed them, as evidenced by repeatedly used kill sites — a sign of long-term planning and coordination among hunters, she said.


Recent findings have even indicated Neanderthals were cooking, as evidenced by tiny bits of cooked plant material recovered from their teeth.

Story Source:

The above story is reprinted (with editorial adaptations by ScienceDaily staff) from materials provided by University of Colorado at Boulder.

Journal Reference:

1.                       Wil Roebroeks and Paola Villa. On the earliest evidence for habitual use of fire in Europe. PNAS, March 14, 2011 DOI: 10.1073/pnas.1018116108