Heating-Enabled Formation of Droplet Interface Bilayers Using Escherichia coli Total Lipid Extract

Droplet interface bilayers (DIBs) serve as a convenient platform to study interactions between synthetic lipid membranes and proteins. However, a majority of DIBs have been assembled using a single lipid type, diphytanoylphosphatidylcholine (DPhPC). The work described herein establishes a new method...

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Bibliographic Details
Published in:Langmuir 2015-01, Vol.31 (1), p.325-337
Main Authors: Taylor, Graham J., Sarles, Stephen A.
Format: Article
Language:English
Subjects:
Calorimetry, Differential Scanning
Escherichia coli
Escherichia coli - chemistry
Hot Temperature
Lipid Bilayers - chemistry
Lipids - chemistry
Models, Biological
Surface Properties
Water - chemistry
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Summary:Droplet interface bilayers (DIBs) serve as a convenient platform to study interactions between synthetic lipid membranes and proteins. However, a majority of DIBs have been assembled using a single lipid type, diphytanoylphosphatidylcholine (DPhPC). The work described herein establishes a new method to assemble DIBs using total lipid extract from Escherichia coli (eTLE); it is found that incubating oil-submerged aqueous droplets containing eTLE liposomes at a temperature above the gel–fluid phase transition temperature (T g) promotes monolayer self-assembly that does not occur below T g. Once monolayers are properly assembled via heating, droplets can be directly connected or cooled below T g and then connected to initiate bilayer formation. This outcome contrasts immediate droplet coalescence observed upon contact between nonheated eTLE-infused droplets. Specific capacitance measurements confirm that the interface between droplets containing eTLE lipids is a lipid bilayer with thickness of 29.6 Å at 25 °C in hexadecane. We observe that bilayers formed from eTLE or DPhPC survive cooling and heating between 25 and 50 °C and demonstrate gigaohm (GΩ) membrane resistances at all temperatures tested. Additionally, we study the insertion of alamethicin peptides into both eTLE and DPhPC membranes to understand how lipid composition, temperature, and membrane phase influence ion channel formation. Like in DPhPC bilayers, alamethicin peptides in eTLE exhibit discrete, voltage-dependent gating characterized by multiple open channel conductance levels, though at significantly lower applied voltages. Cyclic voltammetry measurements of macroscopic channel currents confirm that the voltage-dependent conductance of alamethicin channels in eTLE bilayers occurs at lower voltages than in DPhPC bilayers at equivalent peptide concentrations. This result suggests that eTLE membranes, via composition, fluidity, or the presence of subdomains, offer an environment that enhances alamethicin insertion. For both membrane compositions, increasing temperature reduces the lifetimes of single channel gating events and increases the voltage required to cause an exponential increase in channel current. However, the fact that alamethicin insertion in eTLE exhibits significantly greater sensitivity to temperature changes through its T g suggests that membrane phase plays an important role in channel formation. These effects are much less severe in DPhPC, where heating from 25 to 50 °C does
ISSN:0743-7463
1520-5827
DOI:10.1021/la503471m