(Edmonton) Researchers from the Department of Physics and the National Institute for Nanotechnolog are the first to map out the folding pathways of prions.
Prions are malformed proteins that lead to diseases such as Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy (BSE, or "mad cow" disease) in cattle.
A team consisting of researchers from the University of Alberta and the National Institute for Nanotechnology used specially designed optical tweezers to pull apart prion protein molecules and map their motions with unprecedented precision.
"We are using tools from physics to help solve hard problems in biology," says Michael Woodside, a NINT Research Officer with a cross appointment as an assistant professor in Physics. "No-one has ever directly mapped out the folding pathways that are available to the prion protein. By doing this, we get more detailed information about what are the likely states of the protein that should be targeted by therapeutics to prevent the disease."
Most strikingly, Woodside's team found that the unfolded state of the prion may play a key role in the misfolding. "We don't see evidence for the kind of partially-folded, almost native intermediate states that have been postulated to lead to disease. Instead we find evidence for the importance of the unfolded state, which has been more neglected."
The optical tweezers used to pull on the protein molecules were developed by Woodside and his team to measure atomic-scale motions of the molecules in real time as they change structure. The apparatus is situated in NINT, next to the Centennial Centre for Interdisciplinary Sciences (home to the Department of Physics) on the main UAlberta campus.
Two physics students, three physics postdocs and two NINT technicians, along with Woodside, worked on the experiment. The findings were recently published in Proceedings of the National Academy of Sciences USA. The lead authors were Hao Yu, a PhD candidate in Physics, and Dr. Xia Liu, a postdoc who is now a staff scientist at the Canadian Light Source.
Woodside is proud of his team, and predicts the findings will lead to more investigations.
"This is the first study that has directly observed and characterized the formation of non-native (i.e., incorrect) structures in individual protein molecules that contain a single structural domain," he says. "Only a handful of studies have had sufficient spatial and temporal resolution to observe such effects, and they have not seen them."
Woodside adds: "There will be a lot to learn about what makes PrP special, with its unique and peculiar infectiousness, by comparing its folding to the folding of more 'normal' proteins when measured at the same level of precision."
Q & A with Michael Woodside
Do the optical tweezers literally pull apart prion protein molecules?
Yes, think of it as unraveling a ball of yarn, except that the yarn wants to be in a particular shape. We tug on the ends, and the three-dimensional structure unravels into a straight line.
What does the researcher see?
The researcher can't see the individual protein molecule, because it is too small, but he/she can see the plastic beads to which the protein is attached and which we use to apply force-the beads can be seen in the microscope, and you can watch them getting pulled apart as force is applied to the protein. However, the real measurement follows the changes in structure by watching the voltage from the sensitive detectors used to determine precisely the location of the beads.
The paper says, "Our results suggest that the key state for misfolding may be the unfolded state." Does folding occur after the formation of a given protein, or does it occur as the protein is formed?
This is a tricky question. Some folding does occur as the protein is polymerized by the ribosome (called "co-translational folding"), but the extent of this is still being investigated. In the case of the prion protein, the picture is further complicated by the fact that the protein is also passed through a pore in a membrane shortly after it is extruded from the ribosome, which likely unfolds structures that may have formed just after or during translation.
Does the discovery of three misfolding pathways imply that misfolded prions result in three different diseases?
No. In fact, the issue of what connection (if any) there is between we have seen so far and pathogenesis is a key question we are still investigating. It's quite challenging to study this because we can't "freeze" the molecule in a particular shape and use this to cause infection. As it turns out, the diseased state is generally believed to arise from small aggregates consisting of several prion protein (PrP) molecules stuck together. The diseased shape is not thought to be stable for a single protein molecule. Our work is consistent with this picture, since we only ever find the isolated PrP molecules in the correct structure.
An interesting point is that various different kinds of aggregate structures have been observed and/or proposed for PrP. It is still unclear which of these, if any, are related to the disease. It is possible that the multiplicity of aggregates is linked to our observation of multiple misfolding pathways, with different misfolding pathways leading to different aggregate structures. On the other hand, it's also possible that the misfolding pathways of an isolated molecule are not related to the disease, but merely reflect the fact that PrP is conformationally "flexible."
What's the next thing you need to know about prions?
We need to figure out whether and how what we have found is related to formation of the diseased state. This involves looking at the effects of mutations that cause inherited forms of the disease, and also looking at what happens when there is not just one PrP molecule present, but two or more, so that the molecules can aggregate. These are both under way in my lab; the latter in particular is allowing us to explore whether the misfolded states we observed in the isolated PrP molecule are involved in forming structures in aggregates.