Ensslen, T. | bioRxiv Science

bioRxiv2022

A chip-based array for high-resolution fluorescence characterization of free-standing horizontal lipid membranes under voltage clamp

Optical techniques, such as fluorescence microscopy, are of great value in characterizing the structural dynamics of membranes and membrane proteins. A particular challenge is to combine high-resolution optical measurements with high-resolution voltage clamp electrical recordings providing direct information on e.g. single ion channel gating and/or membrane capacitance. Here, we report on a novel chip-based array device which facilitates optical access with water or oil-immersion objectives of high numerical aperture to horizontal free-standing lipid membranes while conrolling membrane voltage and recroding currents using micropatterned Ag/AgCl-electrodes. We demonstrate both wide-field and confocal imaging, as well as time-resolved single photon counting on free-standing membranes spanning sub-picoliter cavities are demonstrated while electrical signals, including single channel activity, are simultaneously acquired. This optically addressable microelectrode cavity array will allow combined electrical-optical studies of membranes and membrane proteins to be performed as a routine experiment.

bioengineering↗

bioRxiv2021

Resolving isomeric posttranslational modifications using a nanopore

Posttranslational modifications (PTMs) of proteins are crucial for cellular function but pose analytical problems, especially in distinguishing chemically identical PTMs at different nearby locations within the same protein. Current methods, such as liquid chromatography-tandem mass spectrometry, are technically tantamount to de novo protein sequencing1. Nanopore techniques may provide a more efficient solution, but applying the concepts of nanopore DNA strand sequencing to proteins still faces fundamental problems2-4. Here, we demonstrate the use of an engineered biological nanopore to differentiate positional isomers resulting from acetylation or methylation of histone protein H4, an important PTM target5,6. In contrast to strand sequencing, we differentiate positional isomers by recording ionic current modulations resulting from the stochastic entrapment of entire peptides in the pores sensing zone, with all residues simultaneously contributing to the electrical signal. Molecular dynamics simulations show that, in this whole-molecule sensing mode, the non-uniform distribution of the electric potential within the nanopore makes the added resistance contributed by a PTM dependent on its precise location on the peptide. Optimization of the pores sensitivity in combination with parallel recording and automated and standardized protein fragmentation may thus provide a simple, label-free, high-throughput analytical platform for identification and quantification of PTMs.

biophysics↗