The degenerative disorder myotrophic lateral sclerosis (also known as ALS or Lou Gehrig’s disease) is commonly associated with a mutated version of the SOD1 gene, encoding the superoxide dismutase-1 enzyme. The prion-like misfolding of SOD1 has been suspected as the cause of the disease, but this folding mechanism remains unclear due to the difficulties in directly observing protein folding. Using optical tweezers, scientists at the University of Alberta have been able to directly investigate how a single molecule of SOD1 folds and unfolds.
To do this, a single molecule of SOD1 protein is held covalently on two ends by optical tweezers. These tweezers attach to the protein via a pair of DNA handles bound to polystyrene beads, which are then controlled using laser beams. By pulling apart the protein from opposite ends, the process of repeated folding and unfolding allows the team to study patterns of protein formation.
"When we pulled the protein apart, we were expecting its structure to come apart all at once based on what was previously known, but what we found instead was a mess," said principal author of the study, Michael Woodside. "But clearer patterns started to emerge after unfolding and refolding it several thousand times. You get a lot more detail working at this single-molecule scale, and it allows us to start piecing the whole picture together."
According to the team’s published findings in Nature, a number of previously undetected intermediate states were observed, which were consistent with the formation of individual beta strands in the native structure. They identified a stable core of the protein that unfolds last and refolds first.
"What we are finding is that when it folds up to an incorrect state, it actually always starts off making the same stable core that you find when it goes into the correct state. It just takes a wrong turn partway down that pathway," said Woodside.
The paper concludes that “partially folded intermediates thus play a crucial role mediating between native and non-native folding.” These findings suggest a mechanistic explanation for SOD1’s tendency for prion-like misfolding, giving insight for possible targets in therapeutic interventions.
"When you don't understand why something is misfolding, it becomes difficult to target therapeutic treatments. So understanding where things are going wrong helps the targeting process become more rational rather than leaning on random screening," said Woodside.