Incorporation of porin channels into miniaturized bilayers Tivadar - - PowerPoint PPT Presentation

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 Small transmembrane domain Add peptide in water solution insertion almost immediate

Amplitude (pA) 20 40 60 80 100 120 Count (N) 0.2 0.4 time (s) 5 10 15 20 25 current (pA) 40 80 120

Hydrophobic porins are different

OmpA in detergent micelle and lipid bilayer (courtesy of Dr. J Bond, University Oxford)

• Membrane proteins with

a significant hydrophobic domain denature in pure water solution.

• During extraction &

purification, detergent is used. 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

OmpA transition from mycelle to bilayer (courtesy of Dr. J Bond, University Oxford)

• I. Add concentrated

micelle solution to bilayer chamber. → Detergent concentration goes far below CMC

• II. Micelles

dissociate, leaving the protein to denature, aggragate, or insert into BLM

• III. By applying

voltage and destabilizing the BLM, a tiny portion

• f proteins will

insert.

Micellar insertion into adsorbed BLM Sadly, this does not work for the glass-adsorbed bilayer! 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. The glass-adsorbed bilayer immediately breaks

• n 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 Detergent removal by Biobeads in the native GUV solution Can insert OmpF, measure gating of protein.

OmpF insertion and gating, measured at -150 mV on the Nanion box

In principle it works: 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)
• II. Charge-charge fusion

negatively charged SUV coupling to positively charged BLM

• III. Phosphocolin-Phosphocolin fusion X

Bilayer:

• PC + 10% -PE

SUV:

• PC + 10% -PE √

√ E.coli polar extract (67% PE) √ √ 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 √ √

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

+75 mV, 150 mM KCl, symmetric salt, DPhPE+DPhPC GUV, DPhPE+DPhPC SUV

Counting OmpF insertions

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.

0.084 μM 118000 0.5 0.166 μM 59000 1 0.326 μM 29500 3 0.770 μM 11800 7 1.412 μM 5900 14 OmpF trimer concentration Lipid / OmpF trimer

• Approx. OmpF /

liposome

“1” “2” “3” “4” “5” “6” “7” “8” “9” “10” 5 10 15 20 25 30 35

0.08 uM

“1” “2” “3” “4” “5” “6” “7” “8” “9” “10” 5 10 15 20 25 30 35

0.166 uM

“1” “2” “3” “4” “5” “6” “7” “8” “9” “10” 5 10 15 20 25

0.33 uM

“1” “2” “3” “4” “5” “6” “7” “8” “9” “10” 5 10 15 20 25

0.77 uM

“1” “2” “3” “4” “5” “6” “7” “8” “9” “10” 5 10 15 20 25 30 35 40 45

1.4 uM

Number of OmpF trimers per insertion (normalized)

Possible to keep the number of insertions constant

• Rapid perfusion
• EDTA
• Extreme dilution

Our version with microfluidic access (perfusion)

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 (Ca2+ / Mg2+ for PE fusion, can be perfused) 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+ –

Comparison et al.

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