ERA NanoSci - Novel force spectroscopy with nanopores
Biological and Biomimetic Chemistry
Final Report Abstract
Small holes in insulating membranes, nanopores, can be used as label-free sensors for biological molecules in solution. At the same time nanopores are ubiquitous building blocks in nature. As nanopores are emerging as powerful single molecule sensors and entering the market for DNA sequencing in the near future, many questions remain concerning the detailed molecular interactions between the translocating molecules and the nanopore’s interior and exterior surfaces. In the Era nanotechnology project NANOPORE the member laboratories developed novel hybrid nanopores, in order to create atomically controlled nanopores for better sensing and novel force spectroscopic techniques. The nanopores based on protein-/solid-state nanopores, made from DNA origami or in graphene layers have the potential to revolutionize nanopore research and technology in the future. The overarching goal of the team in Bremen is to understand the molecular mechanism of channel selectivity and permeation of small molecules. Typical examples are nutrients uptake or permeation of antibiotics across bacterial membrane channels, however to date there are no reliable methods available. Obviously channel controls the permeability by size exclusion but also through the interaction with the channel surface in particular the charge pattern in the constriction zone. To understand these effects we develop experimental techniques. We patched single protein channel reconstituted into giant liposomes. The smaller membrane patch compared to the classical planar lipid bilayer improved the time resolution. Combining nanopipettes with microfluidics allows to work in microliter volumes and the potential for future parallelization. Information on the underlying physics can be obtained by temperature dependent measurements or by variation of the solvent. Surprisingly using a particular ionic liquid (Butyl-methyl imidazolium chloride, BMIM Cl) in aqueous solution resulted in a reduction in channel conductance substantially stronger than expected by an enhanced viscosity. A molecular explanation was given by our collaboration partner Prof. U. Kleinekathöfer using allatom-modelling of the channel conductance. Surprising the presence of BMIMCl reduced substantially the translocation of antibiotics and allows the detection of fast events. A further example was the presence of Mg2+ ions on enrofloxacin permeation through OmpF, the main channel present in the outer cell wall of bacteria, the constriction zone. Molecular modelling in collaboration with the team of Prof. M. Ceccarelli (Cagliari) revealed in this case that enrofloxacin requires a particular orientation to overcome the constriction zone. Mg2+ binds strongly next to the entry and reverses the charge pattern causing several order increase of events. Within this project we improved the instrumental technique and we could demonstrate a number of surprising effects how solvent or physical parameter influence translocation through small nanopores. Furthermore we created a network together with two computer modelling groups. Furthermore we raised the interest for this methodology which allowed us to set a larger consortium with currently implies about 150 researchers dealing with broad aspects of translocation.
Publications
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Permeation through nanochannels: revealing fast kinetics. J Phys Condensed Matter 22 (2010) 454131
Mahendran R K, Pratik RS, Arning J, Stolte S, Kleinekathoefer U, Winterhalter M
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Simple Reconstitution of Protein Pores in Nano Lipid Bilayers. Nano Lett. 11 (2011) 3334-40
Gornall JL, Mahendran KR, Pambos OJ, Steinbock LJ, Otto O, Chimerel C, Winterhalter M, Keyser UF
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Antibiotic Permeation across OmpF Channel: Modulation of the Affinity Site in the Presence of Magnesium. J Phys Chem B. 116 (2012) 4433-8
Singh PR, Ceccarelli M, Lovelle M, Winterhalter M, Mahendran KR
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Protein translocation through TOM40: kinetics of peptide release. Biophys J. 102 (2012) 39-47
Mahendran KR, Romero-Ruiz M, Schlosinger A, Winterhalter M, Nussberger S
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Pulling Peptides across Nano-channels: Resolving Peptide Binding and Translocation through the Hetero-oligomeric Channel from Nocardia farcinica" ACS Nano 6 (2012) 10699- 707
Mahendran KR, Singh P, Bárcena-Uribarri I, Modi N, Kleinekathöfer U, Benz R, Winterhalter M
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Protein reconstitution into free standing planar lipid membranes for electrophysiological characterization. Nature Protocols volume 10, pages 188–198 (2015)
Gutsmann T, Heimburg T, Keyser U, Mahendran RK, Winterhalter M
