DataPDF Available

The Assembly and Use of Tethered Bilayer Lipid Membranes (tBLMs)

DOI:10.1007/978-1-4939-1752-5_4
Authors:
Charles Cranfield at University of Technology Sydney
Charles Cranfield
Sonia Carne
Sonia Carne
Sonia Carne
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Boris Martinac at Victor Chang Cardiac Research Institute
Boris Martinac
Bruce Cornell at Surgical Diagnostics Pty Ltd
Bruce Cornell
  • Surgical Diagnostics Pty Ltd
Download file PDF
Read file
Download citation

Abstract and Figures

Because they are fi rmly held in place, tethered bilayer lipid membranes (tBLMs) are considerably more robust than supported lipid bilayers such as black lipid membranes (BLMs) (Cornell et al. Nature 387(6633): 580–583, 1997). Here we describe the procedures required to assemble and test tethered lipid bilayers that can incorporate various lipid species, peptides, and ion channel proteins. Key words Tethered bilayer lipid membranes , AC impedance spectroscopy , Ion channels , Lipid bilayers
Figures - uploaded by Boris Martinac
Author content
All figure content in this area was uploaded by Boris Martinac
Content may be subject to copyright.
Content uploaded by Boris Martinac
Author content
All content in this area was uploaded by Boris Martinac on Jan 16, 2015
Content may be subject to copyright.
45
Dylan M. Owen (ed.), Methods in Membrane Lipids, Methods in Molecular Biology, vol. 1232,
DOI 10.1007/978-1-4939-1752-5_4, © Springer Science+Business Media New York 2015
Chapter 4
The Assembly and Use of Tethered Bilayer Lipid
Membranes (tBLMs)
Charles Cranfi eld , Sonia Carne , Boris Martinac ,
and Bruce Cornell
Abstract
Because they are fi rmly held in place, tethered bilayer lipid membranes (tBLMs) are considerably more
robust than supported lipid bilayers such as black lipid membranes (BLMs) (Cornell et al. Nature
387(6633): 580–583, 1997). Here we describe the procedures required to assemble and test tethered lipid
bilayers that can incorporate various lipid species, peptides, and ion channel proteins.
Key words Tethered bilayer lipid membranes , AC impedance spectroscopy , Ion channels , Lipid bilayers
1 Introduction
Tethered membranes consist of a metal electrode, typically gold, to
which tethering moieties are anchored preferably via benzyl disul-
fi de groups ( see Note 1 ). The tethers incorporate into one leafl et
of a subsequently self-assembled lipid bilayer, thus tethering the
bilayer to the metal surface [
1 ] (Fig. 1 ). Interspersed with the teth-
ers are spacer molecules that provide a scaffolding for the tether
molecules. When the amphiphilic tethering molecules are inter-
spersed with polar short-chained spacer molecules a volume
between the metal surface and the bilayer is formed that creates an
aqueous reservoir for ions crossing the membrane. Because they
can be fi rmly held in place with a chemical attachment to the metal,
tethered lipid bilayers are far more robust than solvent based
BLMs. The formation of tBLMs is also a more predictable and
controllable process than forming untethered BLMs. tBLMs
remain intact for months, unlike untethered BLMs which typically
have lifetimes of the order of minutes to hours.
The process of depositing smooth (<1.5 nm) ultrapure
(99.9995 %) gold onto polymeric substrates with no contaminating
intermediate metal layers involves considerable process development.
46
The resulting gold patterned electrodes are, however, ideally suited
to the preparation of reproducible, well-sealed tethered mem-
branes. The tethering chemistry is coated from an ethanol solution
onto the gold surface immediately following gold deposition. This
protocol describes the techniques required to use pre-prepared
gold electrodes supplied from the company SDx Tethered
Membranes Pty Ltd (SDx), the unique supplier in the world of such
a tBLM platform. Gold patterned, chemically coated electrodes (as
25 mm × 75 mm slides (Fig.
2a )) are shipped to the user in hermiti-
cally sealed foil packages in ethanol solution. This format is chosen
in order to provide the user the fl exibility of forming tBLMs com-
prising their choice of lipid. The coated electrodes may also be
Fig. 1 Tethered bilayer lipid membrane (tBLM) schematic
Fig. 2 Components required to form a tBLM fl ow cell. ( a ) Electrodes pre-coated with tethering chemistry. ( b ) A
fl ow cell cartridge top. ( c ) Alignment jig for use when attaching the electrode to the fl ow cell cartridge ( d )
Silicon rubber pressure pad used when attaching the electrode to the fl ow cell cartridge ( e ) Aluminum pressure
plate used when attaching the electrode to the fl ow cell cartridge
Charles Cranfi eld et al.
47
stored at 4 °C for >1 year. Different tether:spacer densities are
offered in order to accommodate protein or peptide components
of molecular weights from 1 to 350 kDa within the membrane.
2 Materials
Ready-made SDx electrodes comprise a close-packed array of
2–4 nm strands of ethylene glycol that act as spacers and 4 nm strands
of ethylene glycol terminated with a C20 phytanyl that act as mem-
brane tethers . Typically electrodes are provided that have 10 % tether
molecules and 90 % spacer molecules. Electrodes with this ratio of
tethers to spacers have been designated as T10 electrodes . T10 elec-
trodes create tBLMs that are very stable with little membrane leak-
age, and with the ability to incorporate up to 40 kDa of the membrane
bound fraction of proteins and peptides. Although T1 electrodes
provide greater capacity in the tBLM for molecular weights up to
350 kDa to penetrate the bilayer molecules, the resulting membrane
is less stable. The length of the spacer molecules can also be altered.
Hydrophilic spacers of hydroxyl- terminated lipid chains can stretch
to the inner leafl et of the lipid bilayer membrane, or can extend only
halfway to the inner leafl et (as depicted in Fig.
1 ) in order to create
additional space between the gold tethering electrode and the teth-
ered bilayer in order to accommodate protein loops extending
beyond the membrane surface at the inner leafl et.
The SDx six-channel measurement electrodes are assembled into a
fl ow cell cartridge (Fig.
4a ) which, in turn, plugs into the SDx
tethaPod ™ conductance and capacitance reader (Fig.
4b ). A car-
tridge preparation kit is supplied by SDx which consists of:
● Individually packaged electrodes pre-coated with tethering
chemistry (Fig.
2a )
● A fl ow cell cartridge top on which is coated the gold counter
electrode (Fig.
2b )
● An alignment jig for use when attaching the electrode to the
fl ow-cell cartridge (Fig.
2c )
● A silicon rubber pressure pad used when attaching the elec-
trode to the fl ow cell cartridge (Fig.
2d )
● An aluminum pressure plate used when attaching the electrode
to the fl ow cell cartridge (Fig.
2e )
● A pressure clamp also used when attaching the electrode to the
fl ow cell cartridge (Fig.
3f )
● Also supplied by SDx is a standard membrane forming lipid
mixture that has been optimized to achieve the best electrical
seal which comprises 3 mM ethanolic solution of a 70 %:30 %
2.1 Electrode
Selection
2.2 Cartridge
Preparation Kit
2.3 Solutions
Tethered Bilayer Lipid Membranes
48
mix of diether diphytanyl (C16) phosphatidyl choline:diether
diphatanyl (C16) hydroxyl (mixture designated AM199 by
SDx). Alternative lipid combinations may be employed pro-
vided they are soluble in ethanol at 3 mM concentrations at
room temperature ( see Note 2 ).
● Preferred electrolyte solution such as Phosphate Buffered
Saline (PBS).
3 Methods
1. Remove six-channel electrode from its sealed foil package
using tweezers. Care should be taken not to touch the six gold
regions that will form the gold tethered membrane. The side
of the electrode where “SDX” appears inverted is the up - side
3.1 Preparing
Cartridges
Fig. 4 ( a ) Assembled fl ow cell cartridge with electrodes. The fl ow cell cartridge provides the counter electrode
which is overlayed onto the six tethering electrodes with a 0.1 mm gap for perfusion of reagents and buffer
solutions. ( b ) Assembled fl ow cell cartridge fi tted into a tethaPod™ AC impedance reader
Fig. 3 ( a ) The fl ow cell cartridge attached to the electrode slide on the alignment jig. ( b ) The pressure clamp
used when attaching the electrode to the fl ow cell cartridge
Charles Cranfi eld et al.
49
upon which the gold has been deposited (Fig. 2a ). Allow
2–3 min for any residual ethanol to evaporate ( see Note 3 ).
2. Place electrode into alignment jig so that the inverted “SDX”
on the slide overlays the inverted “SDX” on the alignment jig
(Fig.
2b ). This will ensure the gold electrodes are correctly
oriented.
3. Peel the thin plastic protective cover from the underside of the
fl ow cell cartridge, taking care to leave the 0.1 mm fl ow cell lam-
inate and adhesive layer in place .
4. Place the fl ow cell cartridge over the alignment jig with the
adhesive laminate facing the electrode (Fig.
3a ). The fl ow cell
cartridge should be aligned such that the numbers 1–6 on the
cartridge align with the 1–6 on the alignment jig. Insert
the short end of the fl ow cell cartridge nearest to well 6 into
the matching slot in the alignment jig and lower the cartridge
onto the electrode.
5. Press the silicon rubber pressure pad into the fl ow cell car-
tridge top. Then position the aluminum pressure plate over the
assembly and insert into the pressure clamp.
6. Compress by ¾ of a turn of the knob and leave for at least
1 min (Fig
3b ).
7. Gently remove the assembly from the pressure clamp. Remove
the aluminum pressure plate and silicon pressure pad taking
care to prevent fl ow cell cartridge separating from the
electrode.
8. By gently lifting from the underside of the electrode the
electrode- fl ow cell cartridge assembly can be removed intact
from the alignment jig. The remaining exposed gold surfaces
are not critical to bilayer formation. The functional gold teth-
ering electrodes are protected within the fl ow cell cartridge
assembly (Fig
4a ).
9. Once the cartridge is made it is important to attach a bilayer
lipid membrane to the tethers as soon as practicable (within
1–2 min; see Note 3 ).
When forming a tBLM a solvent exchange method is employed as
it permits the formation of tBLMs at low tethering ratios well
beyond that possible employing liposomal fusion.
1. To well 1 add 8 μL AM199 or desired ethanolic lipid formula-
tion via the circular opening in the cartridge top.
2. Wait 10 s and add 8 μL lipid solution to the second well,
and wait a further 10 s before making additions to each of the
subsequent four wells. A delay of 10 s provides a convenient
3.2 Creating
a Tethered Bilayer
Lipid Membrane Using
Solvent Exchange
Tethered Bilayer Lipid Membranes
50
operational delay to permit each well to be incubated for same
period of time.
3. Let each ethanolic solution of lipid incubate within the wells for
exactly 2 min then rinse with 100 μL of PBS (or desired buffer
solution, see Note 4 ) taking care not to introduce air bubbles
into the fl ow cell chamber as this will damage membrane forma-
tion. Alternative lipid mixtures may require optimisation of
their concentration and assembly times to achieve the highest
possible membrane seal.
4. Rinse with at least 3 × 100 μL aliquots of the PBS. After the fi rst
wash with PBS time is no longer a critical factor. From the res-
ervoir side of the fl ow cell cartridge remove 100 μL of the wash
through solution and then repeat rinses to ensure the removal of
any residual ethanol and lipids. Following formation, the assem-
bled tethered bilayer must remain totally covered with aqueous
solutions to prevent membrane disassociation ( see Note 5 ).
The conductance and capacitance of the tethered membrane may
be measured by inserting the assembled electrode within the fl ow
cell cartridge into a tethaPod™ reader. The reader simplifi es the
interpretation of the AC impedance spectrum and provides a mea-
sure of membrane conductance and capacitance. Typical conduc-
tion values for a freshly formed membrane using AM199 in PBS
are 0.35 ± 0.15 μS and capacitance values of 18 ± 2 nF at room
temperature. The conductance is proportional to the ion fl ux
through the membrane and the capacitance is inversely propor-
tional to the membrane thickness. A signifi cant additional mea-
sure using a tethaPod™ is the Goodness of Fit (GOF). This indicates
the quality of match between the experimental data and a model
of the tethered membrane. GOF values of less than 0.1 indicate a
good match of the data to this simple model, and suggest that the
membrane is homogenous. The user also has the option to employ
their own models to fi t the raw impedance data.
Peptides and proteins such as alamethicin , α-hemolysin , gramicidin
A , and valinomycin will spontaneously insert into pre-formed
tBLMs [
1 – 4 ]. The kinetics of their insertion can be measured by
the time dependency of the increase in conduction. Although there
are no established protocols for inserting ionophores into tBLMs
the following guidelines should be considered:
1. Peptides dissolved in ethanol/buffer or methanol/buffer mix-
tures should be less than 10 % ethanol or methanol and be
thoroughly rinsed to remove any residual solvent.
2. In order to obtain quantitative kinetics it is necessary to employ
a controlled fl ow rate for the introduction of the peptide to the
tBLM. This can be achieved using a syringe pump that couples
to the fl ow cell cartridge (Fig.
5 ).
3.3 Testing
the Bilayer Using
AC Impedance
Spectroscopy
3.4 Incorporation
of Proteins
and Peptides
3.4.1 Spontaneous
Inserting Proteins
and Peptides
Charles Cranfi eld et al.
51
3. In order to model the kinetics of the conduction increase
following peptide or protein insertion, important consider-
ations include:
● The temperature of the solutions being added is equivalent
to the temperature in the tBLM.
● The aggregation state of the spontaneous inserting pep-
tide or protein in the aqueous buffer. The form of the con-
centration versus conduction relationship will report on
the aggregation state of the protein or peptide in the mem-
brane, with a nonlinearity suggesting that a multimeric
form of the peptide or protein is required for conduction.
4. Insertion of proteins and peptides may depend on the need for
specifi c charged lipids being present in the tBLM.
5. The conduction of some peptides, such as gramicidin A , is
selective for cations over anions and so applying a fi xed nega-
tive potential bias to the tethering gold electrode will enhance
conduction. The ability to apply such a bias is available on the
TethaPod™ [
1 ].
6. The insertion of peptides such as alamethicin is catalyzed by
the application of a transmembrane potential which can be
applied to the tBLM [
3 ]
Fig. 5 An example of a syringe pump attachment to control fl ow across the tBLMs
Tethered Bilayer Lipid Membranes
52
For membrane intrinsic proteins and peptides, their insertion
occurs at the time of tBLM formation through their inclusion as a
detergent micelle in the hydrating, membrane forming rinse step
( see Subheading
3.2 , step 3 above). When selecting detergents to
form micelles the general rule is to use high aggregation number
detergents such as Brij 58, CYMAL 5, or DDM. Table
1 lists the
upper tolerable concentration levels for AM199 T10 tBLMs for a
list of common detergents.
4 Notes
1. The use of disulfi des rather than thiols results in a greater
stability of the coating solutions.
2. Lipids that are insoluble in ethanol may be dispersed in
ethanol/methanol mixtures. Care should be taken not to use
solvents, such as chloroform, which will degrade the polycar-
bonate cartridge.
3. Drying of the electrodes will result in adsorption of the tether-
ing chemistry to the gold preventing its incorporation into the
subsequently formed lipid bilayer. For this reason electrodes
should not be left exposed to dry for longer than 2–3 min.
Small droplets of residual ethanol on the electrodes will not
affect the subsequent assembly process.
4. A series of impedance spectroscopy measurements have
demonstrated that 2 min incubation in the lipid ethanolic
solution of AM199 produces the best sealed tBLMs at room
temperature.
5. Following formation of the membrane an adhesive seal can be
applied across the cartridge opening to prevent evaporation
and drying of the sample in the fl ow-cell chamber.
3.4.2 Non-spontaneous
Insertion of Proteins
and Peptides
Table 1
Aggregation numbers as reported by Sigma-Aldrich
Detergent Aggregation number Concentration (μM) Concentration (% w/v)
Brij 58 70 1 0.0001
TWEEN 20 Not reported 4 0.0005
Triton X-100 140 15.5 0.001
DDM 98 50 0.0025
CYMAL-5 47 400 0.02
Charles Cranfi eld et al.
53
Acknowledgments
This work was supported by the Australian Research Council, the
National Health and Medical Research Council of Australia (Grant
1047980). We declare that Bruce Cornell is a shareholder, and
Sonia Carne is an employee, of SDx Tethered Membranes Pty Ltd .
References
1. Cornell BA, Braach-Maksvytis VLB, King LG,
Osman PDJ, Raguse B, Wieczorek L, Pace RJ
(1997) A biosensor that uses ion-channel
switches. Nature 387(6633):580–583
2. Vockenroth IK, Atanasova PP, Jenkins ATA,
Köper I (2008) Incorporation of α-hemolysin in
different tethered bilayer lipid membrane archi-
tectures. Langmuir 24(2):496–502
3. Yin P, Burns CJ, Osman PD, Cornell BA (2003)
A tethered bilayer sensor containing alamethicin
channels and its detection of amiloride based
inhibitors. Biosens Bioelectron 18(4):389–397
4. Raguse B, Braach-Maksvytis V, Cornell BA,
King LG, Osman PD, Pace RJ, Wieczorek L
(1998) Tethered lipid bilayer membranes: for-
mation and ionic reservoir characterization.
Langmuir 14(3):648–659
Tethered Bilayer Lipid Membranes
... Three images per section were captured at 20× magnification using an Olympus BX51 fluorescence microscope with an Olympus DP73 camera at varying exposure times (DAPI: 50 ms; FKBPL: 100 ms; CD31: 100 ms). ImageJ 1.53a was used to calculate the mean greyscale value of the fluorescent intensity of FKBPL and CD31 where values were normalised to the SHAM group as previously described [69,70]. To assess the validity of the immunohistochemistry staining, a negative control containing no primary antibody was used for each staining group. ...
... Gold-coated microscope slides with a monolayer coating of 10% benzyl disulphide eleven-oxygen-ethylene-glycol reservoir linkers with a C20 phytanyl group as 'tethers' and 90% four-oxygen-ethylene-glycol reservoir linkers with a terminal OH group as 'spacers' were purchased from SDx Tethered Membranes Pty Ltd., Sydney, Australia. A lipid bilayer was then anchored to the slides using a solvent-exchange technique that employed 3 mM ethanolic solutions of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids Inc., Alabaster, AL, USA) [70]. The solvent used for the exchange was 100 mM NaCl 10 mM Tris buffer at pH 7. Dilutions of the EAC used this same buffer. ...
E-Cigarette Aerosol Condensate Leads to Impaired Coronary Endothelial Cell Health and Restricted Angiogenesis
Article
Full-text available
Cardiovascular disease (CVD) is a leading cause of mortality worldwide, with cigarette smoking being a major preventable risk factor. Smoking cessation can be difficult due to the addictive nature of nicotine and the withdrawal symptoms following cessation. Electronic cigarettes (e-Cigs) have emerged as an alternative smoking cessation device, which has been increasingly used by non-smokers; however, the cardiovascular effects surrounding the use of e-Cigs remains unclear. This study aimed to investigate the effects of e-Cig aerosol condensate (EAC) (0 mg and 18 mg nicotine) in vitro on human coronary artery endothelial cells (HCAEC) and in vivo on the cardiovascular system using a mouse model of ‘e-vaping’. In vitro results show a decrease in cell viability of HCAEC when exposed to EAC either directly or after exposure to conditioned lung cell media (p < 0.05 vs. control). Reactive oxygen species were increased in HCAEC when exposed to EAC directly or after exposure to conditioned lung cell media (p < 0.0001 vs. control). ICAM-1 protein expression levels were increased after exposure to conditioned lung cell media (18 mg vs. control, p < 0.01). Ex vivo results show an increase in the mRNA levels of anti-angiogenic marker, FKBPL (p < 0.05 vs. sham), and endothelial cell adhesion molecule involved in barrier function, ICAM-1 (p < 0.05 vs. sham) in murine hearts following exposure to electronic cigarette aerosol treatment containing a higher amount of nicotine. Immunohistochemistry also revealed an upregulation of FKBPL and ICAM-1 protein expression levels. This study showed that despite e-Cigs being widely used for tobacco smoking cessation, these can negatively impact endothelial cell health with a potential to lead to the development of cardiovascular disease.
View
... Afterwards, the complete lipid bilayer architecture of the tBLM platform was formed by incubating the monolayer-functionalized gold electrode surface with an ethanolic solution of dissolved 70% zwitterionic C20 diphytanyl-dietherphosphatidylcholine lipid and 30% C20 glycerol diphytanyl ether (DPEPC) lipid. A buffer rinsing step was performed in a solvent-exchange type process [40] in order to induce a phase transition of the lipid molecules whereby they self-assemble with one another along with the attached tethered lipids to form a full-spanning tBLM across the gold electrode surface, which includes an ionic reservoir between the gold surface and lower lipid leaflet. To investigate the membrane-disruptive effects of these compounds, electrochemical impedance spectroscopy (EIS) measurements were conducted using tethered bilayer lipid membrane (tBLM) platforms. ...
... Afterwards, the complete lipid bilayer architecture of the tBLM platform was formed by incubating the monolayer-functionalized gold electrode surface with an ethanolic solution of dissolved 70% zwitterionic C20 diphytanyldiether-phosphatidylcholine lipid and 30% C20 glycerol diphytanyl ether (DPEPC) lipid. A buffer rinsing step was performed in a solvent-exchange type process [40] in order to induce a phase transition of the lipid molecules whereby they self-assemble with one another along with the attached tethered lipids to form a full-spanning tBLM across the gold electrode surface, which includes an ionic reservoir between the gold surface and lower lipid leaflet. ...
Mechanistic Evaluation of Antimicrobial Lipid Interactions with Tethered Lipid Bilayers by Electrochemical Impedance Spectroscopy
Article
Full-text available
There is extensive interest in developing real-time biosensing strategies to characterize the membrane-disruptive properties of antimicrobial lipids and surfactants. Currently used biosensing strategies mainly focus on tracking membrane morphological changes such as budding and tubule formation, while there is an outstanding need to develop a label-free biosensing strategy to directly evaluate the molecular-level mechanistic details by which antimicrobial lipids and surfactants disrupt lipid membranes. Herein, using electrochemical impedance spectroscopy (EIS), we conducted label-free biosensing measurements to track the real-time interactions between three representative compounds—glycerol monolaurate (GML), lauric acid (LA), and sodium dodecyl sulfate (SDS)—and a tethered bilayer lipid membrane (tBLM) platform. The EIS measurements verified that all three compounds are mainly active above their respective critical micelle concentration (CMC) values, while also revealing that GML induces irreversible membrane damage whereas the membrane-disruptive effects of LA are largely reversible. In addition, SDS micelles caused membrane solubilization, while SDS monomers still caused membrane defect formation, shedding light on how antimicrobial lipids and surfactants can be active in, not only micellar form, but also as monomers in some cases. These findings expand our mechanistic knowledge of how antimicrobial lipids and surfactants disrupt lipid membranes and demonstrate the analytical merits of utilizing the EIS sensing approach to comparatively evaluate membrane-disruptive antimicrobial compounds.
View
... To measure how the VR18 peptide interacts with lipid bilayers, sparsely tethered bilayer lipid membranes (stBLMs) in conjunction with swept frequency electrical impedance spectroscopy (EIS) was used. 21,22 Changes in membrane conduction and membrane capacitance were J o u r n a l P r e -p r o o f measured in the presence of increasing concentrations of the VR18 peptide on membranes containing either three parts 1-palmitoyl-2-oleoyl-glycero-3-phosphotidylethanolomine (POPE) with 1 part 1-palmitoyl-2-oleoyl-glycero-3-phosphotidyleglycerol (POPG); or as a 60% palmitoyl-2-oleoyl-glycero-3-phosphotidylcholine (POPC) mixture with 40% cholesterol (Avanti Lipids, USA). 23 To create the stBLMs, a first layer of anchoring lipids consisting of tethered benzyl-disulfide (tetra-ethyleneglycol) n = 2 C20-phytanyl 'tethers' with benzyl-disulfide-tetra-ethyleneglycol-OH 'spacers' in the ratio of 1:10 (SDx Tethered Membranes Pty Ltd, Australia) was incubated with gold patterned polycarbonate slides for one hour. ...
... In order to gain insight of the lipid-peptide interaction from the electrical properties of the two different membrane models with the VR18, a further analysis was done using sparsely tethered bilayer lipid membranes (stBLMs) in conjunction with electrical impedance spectroscopy. 22 As a Gram-negative bacterium, P. aeruginosa has a cytoplasmic membrane with a symmetric phospholipid bilayer and an asymmetric outer membrane with an inner surface of phospholipids and an outer layer that incorporates lipopolysaccharide (LPS), creating a permeation barrier. ...
A rationally designed synthetic antimicrobial peptide against Pseudomonas-associated corneal keratitis: Structure-function correlation
Article
Contact lens wearers are at an increased risk of developing Pseudomonas-associated corneal keratitis, which can lead to a host of serious ocular complications. Despite the use of topical antibiotics, ocular infections remain a major clinical problem, and a strategy to avoid Pseudomonas-associated microbial keratitis is urgently required. The hybrid peptide VR18 (VARGWGRKCPLFGKNKSR) was designed to have enhanced antimicrobial properties in the fight against Pseudomonas-induced microbial keratitis, including contact lens-related keratitis. In this paper, VR18's modes of action against Pseudomonas membranes were shown by live cell Raman spectroscopy, live cell NMR, live-cell fluorescence microscopy and measures taken using sparsely tethered bilayer lipid membrane bacterial models to be via a bacterial-specific membrane disruption mechanism. The high affinity and selectivity of the peptide were then demonstrated using in vivo, in vitro and ex vivo models of Pseudomonas infection. The extensive data presented in this work suggests that topical employment of the VR18 peptide would be a potent therapeutic agent for the prevention or remedy of Pseudomonas-associated microbial keratitis.
View
... The distal fraction of the tethers incorporates hydrophobic moieties into the inner leaflet of a lipid bilayer [1]. Due to spacer molecules positioned between the tethering molecules, only a minority of lipids in the inner leaflet are immobilized, while the majority of lipids in both leaflets remain mobile [2] (Fig. 1a). This allows tBLMs to mimic the fluidity of cell membranes [3] and peptides have sufficient space to form ion channels (Fig. 1b). ...
... 1. For gold electrodes with T10 tethering chemistries, peptides and proteins with a membrane-bound fraction up to 40 kD can be incorporated [2]. ...
Determining the Pore Size of Multimeric Peptide Ion Channels Using Cation Conductance Measures of Tethered Bilayer Lipid Membranes
Chapter
  • Jan 2022
Swept frequency electrical impedance spectroscopy (EIS) can be used in conjunction with tethered bilayer lipid membranes to monitor the membrane permeability of ions in real-time (Deplazes et al. J Phys Chem Lett 11:6353–6358, 2020). Conductance readings, as determined by EIS, are a measure of the ability of ions to be transported across membranes. Recording the change in conductance as a function of cation concentration and a comparison between a range of cations permits conclusions to be made about the specificity of cation transport through pores. An estimate for upper pore size and cation selectivity of ion channels can be established using this method.
View
... This surface functionalization scheme is suitable for preparing tBLM platforms according to the manufacturer's protocol. Specifically, the gold electrode slide was first rinsed with ethanol and partially dried before being mounted to the tethaPlate measurement chamber (SDx Tethered Membranes) that contained six flow cells (see also ref. [89]). A 3 mM lipid solution in ethanol of the desired lipid composition was prepared and an 8 µL aliquot was introduced into each flow cell, followed by 3 × 100 µL PBS buffer rinsing steps to each flow channel using the solvent-exchange technique, as previously described [41]. ...
Biophysical Characterization of LTX-315 Anticancer Peptide Interactions with Model Membrane Platforms: Effect of Membrane Surface Charge
Article
Full-text available
LTX-315 is a clinical-stage, anticancer peptide therapeutic that disrupts cancer cell membranes. Existing mechanistic knowledge about LTX-315 has been obtained from cell-based biological assays, and there is an outstanding need to directly characterize the corresponding membrane-peptide interactions from a biophysical perspective. Herein, we investigated the membrane-disruptive properties of the LTX-315 peptide using three cell-membrane-mimicking membrane platforms on solid supports, namely the supported lipid bilayer, intact vesicle adlayer, and tethered lipid bilayer, in combination with quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) measurements. The results showed that the cationic LTX-315 peptide selectively disrupted negatively charged phospholipid membranes to a greater extent than zwitterionic or positively charged phospholipid membranes, whereby electrostatic interactions were the main factor to influence peptide attachment and membrane curvature was a secondary factor. Of note, the EIS measurements showed that the LTX-315 peptide extensively and irreversibly permeabilized negatively charged, tethered lipid bilayers that contained high phosphatidylserine lipid levels representative of the outer leaflet of cancer cell membranes, while circular dichroism (CD) spectroscopy experiments indicated that the LTX-315 peptide was structureless and the corresponding membrane-disruptive interactions did not involve peptide conformational changes. Dynamic light scattering (DLS) measurements further verified that the LTX-315 peptide selectively caused irreversible disruption of negatively charged lipid vesicles. Together, our findings demonstrate that the LTX-315 peptide preferentially disrupts negatively charged phospholipid membranes in an irreversible manner, which reinforces its potential as an emerging cancer immunotherapy and offers a biophysical framework to guide future peptide engineering efforts.
View
View
The role of ion-lipid interactions and lipid packing in ion-induced pores and transient defects caused by phenolic compounds
Article
The transient disruption of membranes for the passive permeation of ions or small molecules is a complex process relevant to understanding physiological processes and biotechnology applications. Phenolic compounds are widely studied for their antioxidant and antimicrobial properties, and many biological activities of phenolic compounds are based on their interactions with membranes. Ions are ubiquitous in cells and are known to alter the structure of phospholipid bilayers. Yet, ion-lipid interactions are usually ignored when studying the membrane-altering properties of phenolic compounds. This study aims to assess the role of Ca²⁺ ions on the membrane-disrupting activity of two phenolic acids and to highlight the role of local changes in lipid packing in forming transient defects or pores. Results from tethered lipid membrane (tBLM) electrical impedance spectroscopy (EIS) experiments showed that Ca²⁺ significantly reduces membrane disruption by CAME and CAF. As phenolic acids are known metal chelators, we used UV-vis and fluorescence spectroscopy to exclude the possibility that Ca²⁺ interferes with membrane disruption by binding to the phenolic compound and subsequently preventing membrane binding. Molecular Dynamics simulations showed that Ca²⁺ but not CAME or CAF increases lipid packing in POPC bilayers. The combined data confirm that Ca²⁺ reduces the membrane-disrupting activity of the phenolic compounds and that Ca²⁺-induced changes to lipid packing govern this effect. We discuss our data in the context of ion-induced pores and transient defects and how lipid packing affects membrane disruption by small molecules.
View
Subtle Changes in pH Affect the Packing and Robustness of Fatty Acid Bilayers
Article
  • Apr 2022
Connecting molecular interactions to emergent properties is a goal of physical chemistry, self-assembly, and soft matter science. We show that for fatty acid bilayers, vesicle rupture tension, and permeability to water and ions are coupled to pH via alterations to lipid packing. A change in pH of one, for example, can halve the rupture tension of oleic acid membranes, an effect that is comparable to increasing lipid unsaturation in phospholipid systems. We use both experiments and molecular dynamics simulations to reveal that a subtle increase in pH can lead to increased water penetration, ion permeability, pore formation rates, and membrane disorder. For changes in membrane water content, oleic acid membranes appear to be more than a million times more sensitive to protons than to sodium ions. The work has implications for systems in which fatty acids are likely to be found, for example in the primitive cells on early Earth, biological membranes especially during digestion, and other biomaterials.
View
Preparing Ion Channel Switch Membrane-Based Biosensors
Chapter
  • Jan 2022
Monitoring the changes in membrane conductance using electrical impedance spectroscopy is the platform of membrane-based biosensors in order to detect a specific target molecule. These biosensors represent the amalgamation of an electrical conductor such as gold and a chemically tethered bilayer lipid membrane with specific incorporated ion channels such as gramicidin-A that is further functionalized with detector molecules of interest.
View
Measuring Activation Energies for Ion Transport Using Tethered Bilayer Lipid Membranes (tBLMs)
Chapter
  • Jan 2022
Model lipid bilayers tethered to a gold substrate with molecular tethers are constructed. The conductance versus temperature dependence curve is then obtained. Here, a method to measure the activation energy for translocation of an ion through existing transmembrane pores in a sparsely tethered bilayer lipid membranes is presented.
View
Tethered Lipid Bilayer Membranes: Formation and Ionic Reservoir Characterization
Article
  • Jan 1998
Using novel synthetic lipids, a tethered bilayer membrane (tBLM) was formed onto a gold electrode such that a well-defined ionic reservoir exists between the gold surface and the bilayer membrane. Self-assembled monolayers of reservoir-forming lipids were first adsorbed onto the gold surface using gold−sulfur interactions, followed by the formation of the tBLM using the self-assembly properties of phosphatidylcholine-based lipids in aqueous solution. The properties of the tBLM were investigated by impedance spectroscopy. The capacitance of the tBLM indicated the formation of bilayer membranes of comparable thickness to solvent-free black (or bilayer) lipid membranes (BLM). The ionic sealing ability was comparable to those of classical BLMs. The function of the ionic reservoir was investigated using the potassium-specific ionophore valinomycin. Increasing the size of the reservoir by increasing the length of the hydrophilic region of the reservoir lipid or laterally spacing the reservoir lipid results in an improved ionic reservoir. Imposition of a dc bias voltage during the measurement of the impedance spectrum affected the conductivity of the tBLM. The conductivity and specificity of the valinomycin were comparable to those seen in a classical BLM.
View
A biosensor that uses ion-channel switches
Article
  • Jul 1997
Biosensors are molecular sensors that combine a biological recognition mechanism with a physical transduction technique. They provide a new class of inexpensive, portable instrument that permit sophisticated analytical measurements to be undertaken rapidly at decentralized locations. However, the adoption of biosensors for practical applications other than the measurement of blood glucose is currently limited by the expense, insensitivity and inflexibility of the available transduction methods. Here we describe the development of a biosensing technique in which the conductance of a population of molecular ion channels is switched by the recognition event. The approach mimics biological sensory functions and can be used with most types of receptor, including antibodies and nucleotides. The technique is very flexible and even in its simplest form it is sensitive to picomolar concentrations of proteins. The sensor is essentially an impedance element whose dimensions can readily be reduced to become an integral component of a microelectronic circuit. It may be used in a wide range of applications and in complex media, including blood. These uses might include cell typing, the detection of large proteins, viruses, antibodies, DNA, electrolytes, drugs, pesticides and other low-molecular-weight compounds.
View
A tethered bilayer sensor containing alamethicin channels and its detection of amiloride based inhibitors
Article
Alamethicin, a small transmembrane peptide, inserts into a tethered bilayer membrane (tBLM) to form ion channels, which we have investigated using electrical impedance spectroscopy. The number of channels formed is dependent on the incubation time, concentration of the alamethicin and the application of DC voltage. The properties of the ion channels when formed in tethered bilayers are similar to those for such channels assembled into black lipid membranes (BLMs). Furthermore, amiloride and certain analogs can inhibit the channel pores, formed in the tBLMs. The potency and concentration of the inhibitors can be determined by measuring the change of impedance. Our work illustrates the possibility of using a synthetic tBLM for the study of small peptide voltage dependent ion channels. A potential application of such a device is as a screening tool in drug discovery processes.
View
Incorporation of ??-Hemolysin in Different Tethered Bilayer Lipid Membrane Architectures
Article
  • Feb 2008
Tethered bilayer lipid membranes are stable solid supported model membrane systems. They can be used to investigate the incorporation and function of membrane proteins. In order to study ion translocation mediated via incorporated proteins, insulating membranes are necessary. The architecture of the membrane can have an important effect on both the electrical properties of the lipid bilayer as well as on the possibility to functionally host proteins. Alpha-hemolysin pores have been functionally incorporated into a tethered bilayer lipid membrane coupled to a gold electrode. The protein incorporation has been monitored optically and electrically and the influence of the molecular structure of the anchor lipids on the insertion properties has been investigated.
View

Linked Research

Transient Potential Gradients and Impedance Measures of Tethered Bilayer Lipid Membranes: Pore-Forming Peptide Insertion and the Effect of Electroporation
Article
January 2014
Charles Cranfield · Bruce Cornell · Stephan L Grage · Paul Duckworth · Boris Martinac