Interaction of pore-forming proteins with biological and synthetic polymers
The use of pore forming proteins (PFPs, aka biological nanopores) as electrophysiological single molecule sensors goes back to pioneering experiments in the decade spanning the turn of the millennium.1-3 In 2010 DNA-stand sequencing by nanopore was first shown by an academic group4 and since 2014 commercial devices are available. The Behrends lab first used nanopore-based single molecule ‘sizing’ with the staphylococcal alpha-hemolysin pore3 to benchmark the microelectrode cavity array,5 the first multichannel planar bilayer chip.6 Later, we found that the pore formed by aerolysin, a toxin produced by another bacterium, is an even better sensor for this application.7 Most recently, we were able to show that the current through this nanopore -and, especially, an engineered variant- is actually sensitive to molecular shape, allowing us to detect very subtle chemical differences due to isomeric posttranslational modification of peptides.8
We are currently engaged in two directions:
- exploring the physicochemical basis of the exquisite sensitivity of nanopore current to molecular properties and
- applying this principle to real-world diagnostic problems and to protein sequencing. The latter route, especially, will be pursued in close collaboration with partners in the framework of the Cluster4Future nanodiagBW.
1: Kasianowicz, J.J., E. Brandin, D. Branton, and D.W. Deamer. 1996. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl. Acad. Sci. U S A. 93:13770–13773. doi:10.1073/pnas.93.24.13770
2: Krasilnikov, O.V., C. Rodrigues, and S. Bezrukov. 2006. Single Polymer Molecules in a Protein Nanopore in the Limit of a Strong Polymer-Pore Attraction. Phys. Rev. Lett. 97:018301. doi:10.1103/PhysRevLett.97.018301.
3: Robertson, J.W.F., C.G. Rodrigues, V.M. Stanford, K.A. Rubinson, O.V. Krasilnikov, and J.J. Kasianowicz. 2007. Single-molecule mass spectrometry in solution using a solitary nanopore. Proc. Natl. Acad. Sci. U S A. 104:8207–8211. doi:10.1073/pnas.0611085104.
4: Derrington, I.M., T.Z. Butler, M.D. Collins, E. Manrao, M. Pavlenok, M. Niederweis, and J.H. Gundlach. 2010. Nanopore DNA sequencing with MspA. Proc. Natl. Acad. Sci. U S A. 107:16060–16065. doi:10.1073/pnas.1001831107.
5: Baaken, G., N. Ankri, A.-K. Schuler, J. Rühe, and J.C. Behrends. 2011. Nanopore-Based Single-Molecule Mass Spectrometry on a Lipid Membrane Microarray. ACS Nano. 5:8080–8088. doi:10.1021/nn202670z.
6: Baaken, G., M. Sondermann, C. Schlemmer, J. Rühe, and J.C. Behrends. 2008. Planar microelectrode-cavity array for high-resolution and parallel electrical recording of membrane ionic currents. Lab Chip. 8:938–944. doi:10.1039/b800431e
7: Baaken, G., I. Halimeh, L. Bacri, J. Pelta, A. Oukhaled, and J.C. Behrends. 2015. High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore. ACS Nano. 9:6443–6449. doi:10.1021/acsnano.5b02096.
8: Ensslen, T., K. Sarthak, A. Aksimentiev, and J.C. Behrends. 2022. Resolving Isomeric Posttranslational Modifications Using a Biological Nanopore as a Sensor of Molecular Shape. J. Am. Chem. Soc. 144:16060–16068. doi:10.1021/jacs.2c06211.
Simultaneous optical and electrical analysis of membrane ion channels and pores
“The holy grail of ion channel studies is to produce an atomic scale movie of an ion channel at work, simultaneously observing conformational and electrical changes as ions flow through the protein.” This quotation from Paul Selvin’s 2003 “New and Notable” comment1 on an early step in that direction (to which the Behprends lab contributed)2 continues to inspire our long-term goal of combining single molecule optical-electrical analysis of the movement and function of membrane proteins and/or of their interaction with ligands and blockers. As we have recently solved many of the difficult problems of experimental apparatus3 and have gained and assembled the necessary experience and competences regarding optics, protein engineering, purification and reconstitution, we are hopeful of a breakthrough in the next few years.
1: Selvin, P.R. 2003. Lighting up single ion channels. Biophys. J. 84:1–2. doi:10.1016/S0006-3495(03)74827-9.
2: Borisenko, V., T. Lougheed, J. Hesse, E. Füreder-Kitzmuller, N. Fertig, J.C. Behrends, G.A. Woolley, and G.J. Schutz. 2003. Simultaneous optical and electrical recording of single gramicidin channels. Biophys. J. 84:612–622. doi:10.1016/S0006-3495(03)74881-4.
3: Ensslen, T., and J.C. Behrends. 2022. A chip-based array for high-resolution fluorescence characterization of free-standing horizontal lipid membranes under voltage clamp. Lab Chip. 22:2902–2910. doi:10.1039/D2LC00357K.
Pushing the boundaries of resolution of voltage-clamp analysis of membrane currents
Not least among our aims in the development of “patch clamp on a chip”, where the electrolyte-filled glass pipette used in patch clamping was replaced by an aperture in a planar glass substrate1 was minimizing electrical measurement noise by reducing stray capacitance through further miniaturization of the recording arrangement.2,3
Moreover, miniaturization of the active electrode, as in our microelectrode cavity array (MECA)4 allows the construction of devices integrating membrane, electrolyte, electrode and CMOS-ASIC amplifier microelectronics, as was first shown by Rosenstein et al. in 2013. Since several years,6 we work with the Chair of Microelectronics in Freiburg, the Hahn-Schickard-Institute for Micro- and Information Technology and the Behrends lab start-up Ionera Technologies to come up with our own bespoke 4-channel version of such a device, which we are currently trying out in the lab.
Its features are optimized for high-resolution, low noise recordings from biological nanopores and ion channels and we are having lot’s of fun with it. Results will be communicated soon.
1: Fertig, N., R.H. Blick, and J.C. Behrends. 2002. Whole cell patch clamp recording performed on a planar glass chip. Biophys. J. 82:3056–3062. doi:10.1016/S0006-3495(02)75646-4
2: Sigworth, F.J., and K.G. Klemic. 2005. Microchip Technology in Ion-Channel Research. IEEE Trans. NanoBiosci. 4:121–127. doi:10.1109/TNB.2004.842471.
3: Sondermann, M., M. George, N. Fertig, and J.C. Behrends. 2006. High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formation. Biochimica et Biophysica Acta (BBA) – Biomembranes. 1758:545–551. doi:10.1016/j.bbamem.2006.03.023
4: Baaken, G., M. Sondermann, C. Schlemmer, J. Rühe, and J.C. Behrends. 2008. Planar microelectrode-cavity array for high-resolution and parallel electrical recording of membrane ionic currents. Lab Chip. 8:938–944. doi:10.1039/b800431e.
5: Rosenstein, J.K., S. Ramakrishnan, J. Roseman, and K.L. Shepard. 2013. Single Ion Channel Recordings with CMOS-Anchored Lipid Membranes. Nano Lett. 13:2682–2686. doi:10.1021/nl400822r.
6: Amayreh, M., G. Baaken, J.C. Behrends, and Y. Manoli. 2019. A Fully Integrated Current-Mode Continuous-Time Delta-Sigma Modulator for Biological Nanopore Read Out. IEEE Trans. Biomed. Circuits Syst. 13:225–236. doi:10.1109/TBCAS.2018.2889536..