Incorporation of porin channels into miniaturized bilayers Tivadar Mach, Mohammed Kreir, Niels Fertig, Mathias Winterhalter Marseille 11 April 2008
Folded classical bilayer Main issues: time resolution (30-50 μs rock bottom) chamber volume (antibiotic concentrations mm ) manual operation
Folded classical bilayer Main issues: time resolution (30-50 μs rock bottom) chamber volume (antibiotic concentrations mm ) manual operation
Miniaturized bilayer
Insertion of hydrophilic peptides Alpha-Haemolysin hydrophilic, water-soluble current (pa) 120 80 40 0 5 10 15 20 25 time (s) Small transmembrane domain Count (N) Add peptide in water solution insertion almost immediate 0.4 0.2 0 0 20 40 60 80 Amplitude (pa) 100 120
Hydrophobic porins are different Membrane proteins with a significant hydrophobic domain denature in pure water solution. During extraction & purification, detergent is used. OmpA in detergent micelle and lipid bilayer (courtesy of Dr. J Bond, University Oxford) This can be an advantage! Insertion of proteins in micelles into conventional bilayer by going below the CMC, forcing proteins into BLM.
Insertion from micelles into Montal-Muller BLM I. Add concentrated micelle solution to bilayer chamber. Detergent concentration goes far below CMC OmpA transition from mycelle to bilayer (courtesy of Dr. J Bond, University Oxford) II. Micelles dissociate, leaving the protein to denature, aggragate, or insert into BLM III. By applying voltage and destabilizing the BLM, a tiny portion of proteins will insert.
Micellar insertion into adsorbed BLM Advantages of micelle-insertion: Simple! Protein is usually purified in detergent, no other steps necessary. Good control on number of proteins inserted when destabilizing voltage is stopped, insertions (usually) stop. Sadly, this does not work for the glass-adsorbed bilayer! The glass-adsorbed bilayer immediately breaks on contact with the micelle solution. Controls detergent sensitive (Down to 1 ppb!)
Alternative insertions into adsorbed BLM Aim: reduce detergent, increase stability. Routes: I. Change lipid composition, pre-dilute protein solution micellar insertion II. Insert protein into pre-formed GUVs before adsorption, make bilayer with proteo-guvs III. Insert protein into SUVs, fuse proteo-liposomes to already standing adsorbed bilayer
Adsorption of proteo-guvs In principle it works: Detergent removal by Biobeads in the native GUV solution OmpF insertion and gating, measured at -150 mv on the Nanion box Can insert OmpF, measure gating of protein. Needed a significant amount of optimization Mohammed
Alternative insertions into adsorbed BLM Aim: reduce detergent, increase stability. Routes: I. Change lipid composition, pre-dilute protein solution micellar insertion II. Insert protein into pre-formed GUVs before adsorption, make bilayer with proteo-guvs III. Insert protein into SUVs, fuse proteo-liposomes to already standing adsorbed bilayer
Proteoliposome (SUV) fusion Fusion strategies: I. Ethanolamine-Ethanolamine fusion (Ca2+ or Mg2+ mediated) Bilayer: -PC + 10% -PE SUV: -PC + 10% -PE E.coli polar extract (67% PE) II. Charge-charge fusion negatively charged SUV coupling to positively charged BLM Bilayer: Stearylamine in -PC bilayer (up to 10-12%). SUV: -PC + 5% -PS E.coli polar extract (23% PG) E.coli polar extract (23% PG) + 5% -PS III. Phosphocolin-Phosphocolin fusion X
Preparation of proteo-suvs Lipid film rehydration sonication (or extrusion) Protein addition after liposome formation (vs. protein addition to lipid film prior to rehydration) adsorbed BLM found to be more stable orientation Protein addition Detergent removal Biobeads, 2 exchanges Dialysis
Do we get fusion? -PE -PE fusion +150 mv Charge-charge fusion -150 mv Short answer: yes
A closer look at fusion -75 mv, 150 mm KCl, symmetric salt, DPhPE+DPhPC GUV, DPhPE+DPhPC SUV
A closer look at fusion Counting OmpF insertions +75 mv, 150 mm KCl, symmetric salt, DPhPE+DPhPC GUV, DPhPE+DPhPC SUV
Controls insertion by liposome fusion Tests whether protein insertion actually occurred by fusion: I. Same treatment of protein solution without liposomes results in no insertions. II. Increases in conductance are quantized by protein content of liposomes, dependent on initial protein concentration. OmpF trimer concentration 0.084 μm 0.166 μm 0.326 μm 0.770 μm 1.412 μm Lipid / Approx. OmpF / OmpF trimer liposome 118000 0.5 59000 1 29500 3 11800 7 5900 14
Number of OmpF trimers per insertion (normalized) 0.08 um 0.166 um 0.33 um 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7 7 7 8 8 8 9 9 9 10 10 10 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 5 10 5 10 1.4 um 0.77 um 0 0 35 15 20 25 0 5 10 15 20 25 30 35 40 45 15 20 25
Possible to keep the number of insertions constant Rapid perfusion EDTA Extreme dilution
Our version with microfluidic access (perfusion)
Comparison et al. DPhPC + 10 mol % DPhPS DPhPC + 10 mol % DPhPE E. coli polar extract DPhPC DPhPC DPhPC + 10 mol% stearylamine concn gradient concn gradient DPhPC + 5 mol% DPhPE concn gradient + Ca2+ / Mg2+ concn gradient + Ca2+ / Mg2+ Proteo-GUV Liposome fusion Lipid composition positive / neutral almost any Storable 3-5 days Proteo-SUVs: frozen few months Automation Easy? Single-channel insertion Easy (calibrate concentrations) Serious problem (need perfusion) Unknown channel patch-clamp channel inserted from start Gradual ( quantized ) insertion, fast steps Protein consumption Small for one experiment large for series Enormous (compared to BLM) Buffer composition Buffer matters Almost any (Ca / Mg for PE fusion, can be perfused) 2+ 2+
Alternative insertions into adsorbed BLM Aim: reduce detergent, increase stability. Routes: I. Change lipid composition, pre-dilute protein solution micellar insertion II. Insert protein into pre-formed GUVs before adsorption, make bilayer with proteo-guvs III. Insert protein into SUVs, fuse proteo-liposomes to already standing adsorbed bilayer