Stable polymer bilayers for protein channel recordings at high guanidinium chloride concentrations

Abstract

Use of chaotropic reagents is common in biophysical characterization of biomolecules. When the study involves transmembrane protein channels, the stability of the protein channel and supporting bilayer membrane must be considered. In this letter we show that planar bilayers composed of poly(1,2-butadiene)-b-poly(ethylene oxide) diblock copolymer are stable and leakfree at high guanidinium chloride concentrations, in contrast to diphytanoyl phosphatidylcholine bilayers which exhibit deleterious leakage under similar conditions. Further, insertion and functional analysis of channels such as a-hemolysin and MspA are straightforward in these polymer membranes. Finally, we demonstrate that a-hemolysin channels maintain their structural integrity at 2M guanidinium chloride concentrations using blunt DNA hairpins as molecular reporters.

Control of subunit stoichiometry in single-chain MspA nanopores

“…We recently demonstrated that octameric MspA M2 forms channels in bilayers composed of poly(1,2-butadiene)-b-poly(ethylene oxide) (PBD-PEO) polymer (10). PBD-PEO bilayers have a two-to three-fold increased lifetime and are more robust towards chemicals and high voltages than membranes made from biological lipids, and can be used in nanopore sequencing experiments (10). Thus, we also examined the channel properties of scMspA insertions in PBD-PEO bilayers.…”

“…Single sc 8 MspA dt M2 pores were inserted into poly(1,2-butadiene)-b-poly(ethylene oxide) (PBD 11 –PEO 8 ) block-copolymer bilayer membranes with a diameter of 100 µm and bathed in a buffer containing 10 mM Tris (pH 7.5), 1 M KCl and 2 M guanidinium chloride (GdmCl). Based on previous data (10) we used GdmCl in the electrolyte solution because it induces a semi-melting state of DNA hairpins enabling smooth DNA passage through the nanopore at low applied voltage resulting in increased DNA capture rate and decreased trapping time.…”

“…Lipid bilayer experiments to check pore formation by MspA proteins were performed in diphytanoylphosphatidylcholine artificial membranes in a custom made bilayer apparatus as previously described (27) and summarized in SI Appendix . Single-channel recodings and hairpin experiments were done in SU-8 wedge-on-pillar aperture and poly(1,2-butadiene)-b-poly(ethylene oxide) copolymer was used to form a bilayer as described (10). Data were collected at 250 kHz sampling rate and digitized.…”

“…While the concept of nanopore DNA sequencing was developed using the staphylococcal α-hemolysin pore (5), it was demonstrated to be a feasible technology using the Mycobacterium smegmatis porin A (MspA) (6). MspA is the most stable of the known pore proteins resisting denaturation by 2% SDS at 100 °C (7) and is functional in many different membrane-like environments (8-10), an important feature for technical applications (3). We first engineered MspA to translocate DNA (11), showing that MspA can distinguish the four nucleobases (12) and then achieved nanopore sequencing of DNA in combination with a motor enzyme (13).…”

Unidirectional Single-File Transport of Full-Length Proteins Through a Nanopore

“…Our platform uniquely combines a robust geometric aperture that supports high voltage measurements with a bilayer polymer-based membrane that withstands high denaturant concentrations. Incorporating into the bilayer an a-hemolysin nanopore, which maintains its integrity at high GdmCl concentrations 51 , enables steady voltagedriven translocation of proteins through the pore. When a negatively charged tail is present on a protein molecule, end-threading into the pore (from either the N-or C-terminus) is facilitated, but the high GdmCl concentration is essential for the transport of proteins with a constant rate.…”