Single polypeptide detection using a translocon EXP2 nanopore.

DNA sequencing using nanopores has already been achieved and commercialized; the next step in advancing nanopore technology is towards protein sequencing. Although trials have been reported for discriminating the 20 amino acids using biological nanopores and short peptide carriers, it remains challenging. The size compatibility between nanopores and peptides is one of the issues to be addressed. Therefore, exploring biological nanopores that are suitable for peptide sensing is key in achieving amino acid sequence determination. Here, we focus on EXP2, the transmembrane protein of a translocon from malaria parasites, and describe its pore-forming properties in the lipid bilayer. EXP2 mainly formed a nanopore with a diameter of 2.5 nm assembled from 7 monomers. Using the EXP2 nanopore allowed us to detect poly-L-lysine (PLL) at a single-molecule level. Furthermore, the EXP2 nanopore has sufficient resolution to distinguish the difference in molecular weight between two individual PLL, long PLL (Mw: 30,000-70,000) and short PLL (Mw: 10,000). Our results contribute to the accumulation of information for peptide-detectable nanopores.

PV1 Protein from Plasmodium falciparum Exhibits Chaperone-Like Functions and Cooperates with Hsp100s.

Plasmodium falciparum parasitophorous vacuolar protein 1 (PfPV1), a protein unique to malaria parasites, is localized in the parasitophorous vacuolar (PV) and is essential for parasite growth. Previous studies suggested that PfPV1 cooperates with the Plasmodium translocon of exported proteins (PTEX) complex to export various proteins from the PV. However, the structure and function of PfPV1 have not been determined in detail. In this study, we undertook the expression, purification, and characterization of PfPV1. The tetramer appears to be the structural unit of PfPV1. The activity of PfPV1 appears to be similar to that of molecular chaperones, and it may interact with various proteins. PfPV1 could substitute CtHsp40 in the CtHsp104, CtHsp70, and CtHsp40 protein disaggregation systems. Based on these results, we propose a model in which PfPV1 captures various PV proteins and delivers them to PTEX through a specific interaction with HSP101.

Expression and characterization of the Plasmodium translocon of the exported proteins component EXP2.

The malaria parasite Plasmodium falciparum requires the Plasmodium translocon of exported proteins (PTEX) to proliferate in human red blood cells. During the blood stages of malaria, several hundred parasite-encoded proteins are exported from the parasite into the cytosol of red blood cells. PTEX is the translocon for protein export and comprises 5 proteins: EXP2, PTEX150, PTEX88, Hsp101 and TRX2. Among them, EXP2 is thought to constitute the transmembrane pore, whereas the other components seem to play a role in unfolding the luggage proteins or providing a driving force. However, detailed functional and structural characterizations of PTEX proteins have not been performed. In this study, we expressed and characterized the membrane-associated component EXP2. Because expression of EXP2 is lethal to E. coli, EXP2 was expressed as a fusion protein with GST, and the recombinant EXP2 was obtained by protease digestion. The recombinant EXP2 formed pores in bilayer lipid membranes. The inner diameter of the pore was estimated to be approximately 3.5 nm based on electron microscopy images and channel currents. From this size and the molecular mass as determined by size exclusion chromatography and blue native polyacrylamide gel electrophoresis, we determined that the pore comprises approximately 10-12 EXP2 subunits. However, there is a possibility that the pore structure is different in the PTEX complex. These results provide important insights in the protein transport mechanism of PTEX, which will aid in developing new drugs targeting PTEX.