|Fig. 5 from Rey et al article in Molecular & Cellular Proteomics: differential deuteration levels mapped to the available structure of XRCC4(1–200) complexed with the tandem BRCT domains. Click on the image to see a larger version.
How can monkey cups help proteomics? A recent paper in Molecular & Cellular Proteomics describes a special protease derived from this leaf of a carnivorous plant (1). The protease may be useful for determining protein structures and mapping protein interactions, particular those of intrinsically disordered proteins.
Monkey cups are special leaves on an insect-eating plant called nepenthes. These leaves catch water to drown insects. Once a victim has been caught in a cup, the plant secretes digestive juices, which include the protease, into the water to break down the insect and absorb its nutrients. However, rainforest monkeys have caught on that these leaves can work as water reservoirs and drink out them, hence the name “monkey cups.”
David Schriemer’s group at the University of Calgary came across the monkey cup protease when searching for an aspartic protease that was phylogenetically distant from the standard workhorse protease pepsin. The Schriemer group is interested in hydrogen-deuterium exchange mass spectrometry, because it has the potential to reveal high-resolution structural and temporal details about complex protein systems. Proteins can be thought of as breathing: They exchange their hydrogens for deuteriums when bathed in D2O. The hydrogens that get switched for deuteriums help researchers figure out details of protein structure, folding and interactions.
Besides D2O, the method also uses pepsin to cut proteins into manageable pieces. But pepsin has its drawbacks. It isn’t always an efficient protease, and it doesn’t consistently cut all proteins at the right spots. “We want 100 percent coverage so we can track exactly where the deuteriums go,” says Schriemer.
So when the investigators came across the monkey cup protease called nepenthesin, they were intrigued by it, because it could perhaps overcome the drawbacks with pepsin. “We were surprised to find that nepenthes extracts are very poorly characterized, and only a handful of studies exist. What evidence there was suggested it was worth a look,” says Schriemer.
The investigators grew a few nepenthes plants in their lab, fed them fruit flies to induce secretion of digestive juices, collected those juices, and isolated the protease.
Schriemer and colleagues then tested nepenthesin in the place of pepsin. They found they could “now look at larger protein complexes and expect to get better sequence coverage,” says Schriemer. The protease “really extends the reach of the method and gets us closer to a proteomics-grade version of the technology.”
In particular, nepenthesin can better handle intrinsically disordered proteins than pepsin, which doesn’t deal well with the prolines and charged residues that often dominate these kinds of proteins.
Schriemer says the next step for the group is to scale up nepenthesin production. “We have many people asking for the enzyme!” says Schriemer.
- 1. Rey, M., et al. Mol. Cell. Proteomics, doi: 10.1074/mcp.M112.025221
Rajendrani Mukhopadhyay (email@example.com) is the senior science writer for ASBMB Today and the technical editor for The Journal of Biological Chemistry. Follow her on Twitter (www.twitter.com/rajmukhop), and read her ASBMB Today blog, Wild Types.