Large and Stable Nanopores Formed by Complement Component 9 for Characterizing Single Folded Proteins.

Abstract

Biological nanopores offer a promising approach for single-molecule analysis of nucleic acids, peptides, and proteins. The work presented here introduces a biological nanopore formed by the self-assembly of complement component 9 (C9). This exceptionally large and cylindrical protein pore is composed of 20 ± 4 monomers of C9 resulting in a diameter of 10 ± 4 nm and an effective pore length of 13 nm. These poly(C9) pores remain stable for up to 30 min without indications of gating, flickering, or clogging across a range of transmembrane voltages (-150 to +150 mV) and ionic strengths (50 to 1000 mM). At physiologic pH, the ring-shaped distribution of negative and positive surface charges in the lumen of the pore enables capture of analyte proteins by electro-osmotic flow and leads to residence times of analyte proteins whose most probable values can exceed 300 μs. We used poly(C9) nanopores to determine the volume and shape of unlabeled folded proteins with molecular weights between 9 and 230 kDa with unprecedented accuracy in the context of resistive pulse recordings. Finally, poly(C9) pores made it possible to distinguish between the open and closed conformations of adenylate kinase based on differences in current modulations within resistive pulses and the corresponding differences in approximations of their shape. Thus, poly(C9) nanopores enable highly sensitive and accurate characterization of a wide range of natively folded proteins on a single molecule level.