Proteins are a critical part of our cells, carrying out and assisting in a vast number of processes including DNA replication, chemical catalysis and transport of essential molecules. They even provide physical structure to our bodies as part of cartilage, hair, and nails. Proteins’ proper function is, consequently, paramount to our most basic natural equilibrium.
When proteins are damaged or folded improperly, enzymes (called proteases) will typically break them down. Some misfolded protein are, unfortunately, resistant to this critical process. Instead, they begin to aggregate into fiber-like structures called amyloid fibrils. This is alarming for two reasons: the misfolded protein impose their improper structure on healthy copies, thereby propagating the error, and the creation of these fibrils is often toxic to the surrounding cells. In fact, amyloid formation is associated with a range of diseases including Alzheimer’s, Parkinson’s, and type 2 diabetes.
Worse still, a subset of amyloid protein can actually transfer to new organisms, much like viruses and bacteria. These are called prions, the most famous of which is likely spongiform bovine encephalopathy, colloquially called Mad Cow Disease. This illness, which leads to rapid mental deterioration, is caused by humans ingesting the misfolded protein through infected beef. After an extended incubation period, the cow prion imposes its structure onto our own protein, resulting in an amyloid aggregation cascade. Compared to virology and bacteriology, the study of amyloid and prion diseases is still a new field. Scientists are searching for fundamental answers regarding behavior and pathology that lie in the specific molecular interaction between infectious prions and healthy protein.
Schematic of potential amyloid behavior for a prion protein (PrP) fragment. The misfolded structure propagates the malignant conformation through hydrogen bonding with normal (wildtype) protein.
How can cryo-EM help?
Cryo-electron microscopy (cryo-EM) allows researchers to visualize protein in their native cellular environment, immobilized and frozen in place. This is essential for prion diseases as the proteins aggregate aggressively outside of the body, transitioning through a variety of structures and states. This means that most analytical techniques, such as NMR and crystallography, are simply too slow to capture important early protein interactions. Today, scientists are using cryo-EM in exciting ways to research these complex systems.
A study by Vázquez-Fernández, et al., performed in collaboration with Thermo Fisher Scientific, obtained structural details for the prion fibrils thanks to cryo-transmission electron microscopy (cryo-TEM). By observing the behavior of a truncated form of the protein a stacked, helix-like (solenoid) motif was clearly visualized. Fragment studies like this are useful for illuminating full protein behavior and revealing potential sites for drug targeting.
Reconstruction of a fibril (red, left) composed of truncated prion protein (PrP27-30). Different colors in the ribbon diagram (right) represent different rungs of the proto-fibril. These model systems, composed of protein fragments, help researchers determine potential interaction sites and models for the full-length protein. Adapted from Vázquez-Fernández, et al. PLoS Pathogens, Sep. 2016, 12(9).
The general difficulty in studying these amyloid protein aggregates lies not only in the speed at which they form but in their original, native structures as well. These are often disordered, which interferes with their crystallization. This means that the rapidly forming intermediate states and the normal, functioning structures evade characterization. The cryo-EM technique of microelectron diffraction (MicroED) can use small nanocrystals of the protein to obtain structural information. MicroED has shown great utility in the overall study of amyloid protein and has also demonstrated success with prion protein fragments.
A collaborative effort between researchers at UCLA and Howard Hughes Medical Institute employed MicroED to analyze two fragments of the tau protein (VQIINK and VQIVYK), a common model system of amyloid aggregation. They were able to determine the structure of VQIINK, which was not possible prior to MicroED, as the fragment did not readily form large enough crystals. Additionally, they were able to use this information to design an inhibitor that targeted the VQIINK region of the full peptide, and had a much greater success at halting the aggregation process than previous inhibitors designed for the other, VQIVYK, region.
Overall, the ongoing study of amyloid protein is important not just because of the known prion diseases, but because infectious protein may be more prevalent than we previously thought. Individuals with one amyloid disease have a greatly increased risk of contracting additional amyloid illnesses, and studies have found that various amyloid protein are capable of aggregating with other amyloid species, despite structural and functional differences. There is a pressing need for a greater understanding of these interrelated disorders, and cryo-EM is ideally suited to advance this field.
Alex Ilitchev, PhD, is a Scientific Content Writer at Thermo Fisher Scientific.