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Investigation of lipid bilayers with naturally
occuring protein pores and biomimmetic
DNA channels
Swati Krishnan and Vera Arnaut
Venue: ZNN, 2nd oor
October 19, 2016
Contents
0.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.1.1 Lipid bilayer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.1.2 Elemental properties of pores . . . . . . . . . . . . . . . . . . . . . . . . . .
0.1.3 Protein channels and functions . . . . . . . . . . . . . . . . . . . . . . . . .
0.1.4 Biomimetic DNA channels . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.2 Experimental protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.2.1 Over view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.2.2 Painted bilayer setup and electrical characterization of channels . . . . . . . .
0.2.3 Giant unilamellar vesicles - Inverted emulsion technique . . . . . . . . . . . .
0.2.4 Modication of the DNA pores with Fluorescentlylabeled staples for imaging .
0.2.5 Fluorescence based detection of hydrophobic interactions between DNA and
lipid membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.3 Investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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0.1 Introduction
0.1.1 Lipid bilayer
Cell membranes are essential boundaries encapsulating the cellular machinery with a lipid bilayer and
several embedded proteins to allow controlled communication with the external environment. A lipid
molecule is an amphiphile with a hydrophillic head and hydrophobic fatty acid tail. In a bilayer the
lipids are arranged in such a way that the hydrophobic tails of both the layers interact with each other.
The edges of the bilayer are not in a favourable state as at the edges the tails are exposed to the
surrounding hydrophillic environment. Based on the geometry of the lipid, they tend to form vesicles
or micelles.
Model membrane systems are made invitro to study membrane processes and for biotechnological
Figure 0.1:
Dierent lipid structures
applications. One of the earliest methods to make model lipid bilayers invitro is the production of
painted bilayers. The lipids are dissolved in an organic solvent like decane and painted across a teon
aperture separating two buer reservoirs (Figure 0.2). The orbit mini system used in our studies is
a modication of the painted bilayer technique. A 2x2 microarray cavity chip on an inert polymer
is used to paint the bilayers with a small brush. Each cavity has a set of electrodes which enables
current recordings. The cavities act as the teon aperture in the classical painted bilayer system.
0.1.2 Elemental properties of pores
Channels or pores allow the movement of molecules across an impermeable membrane. The dimensions
of the pore dictate the number of molecules it will transport across the membrane in a given time.
Nature of the channel like its charge, presence of receptors etc. dictate selectivity of the ions passing
through the pore. The molecules which are smaller than the pore sheds its hydration shell which is
usually compensated by interactions between the pore walls and the molecule. The conductance of a
pore is given by :
γ = A/ρL
(0.1)
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Figure 0.2:
Left:MECA chip with 4 cavities; Right: Painted bilayer technique.
where ρ is the resistivity of the solution, L is the length of the pore and A is the area of the pore.
For a pore with a dened length and area in a certain solution and small test potential equation (0.1)
provides the (maximum) limiting conditions. It can be deduced from the equation that longer channels
are less conductive than shorter counterparts. Once a potential eld is applied, the conductance of
the ion channel can be measured using :
γ = I/V
(0.2)
Consider the following trace The trace is obtained by measuring passage of ions or current across the
Figure 0.3:
Example current trace
bilayer when protein pores are added to it. The otherwise impermeable membrane now shows a small
step wise increases in its conductance. Each step increase is viewed as an addition of a pore to the
membrane and each decrease is the removal of a pore. The length of the step is the mean life time
of the pore in the membrane. Note that at time zero the bilayer already shows conductance which
indicates?. The smaller peaks which are more transient are due to uctuations in the membrane or
pore structure, often called gating events. How can we nd the conductance of a single pore from
this trace? We know from equation (0.2) that γ is I/V. The smallest sustainable jump or the unitary
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conductance step in the trace will give us an indication of I or current across a single pore. Since we
apply the potential across the bilayer we also have the V value. Note the subsequent higher jumps
in the current trace would be approximately a multiple of the smallest step indicating simultaneous
insertion of 2,3 or more pores.
0.1.3 Protein channels and functions
Gramicidin
An ideal model pore would have a precisely dened structure, functionally similar to wide range of
natural pores and should be able to withstand dierent experimental conditions. Gramicidin was the
rst ion selective pore to be crystallized. The availability of structural details as well as functional
robustness sealed the place for Gramicidin as the model pore. Gramicidin is a highly hydrophobic
peptide containing alternating L and D amino acids. Both the N and C terminals of the peptide are
"blocked" meaning having no free charges, making it poorly soluble in water. The chirality of the
amino acids of the linear peptide renders Gramicidin sensitive to its environment. There are two main
folding conformations identied for the peptide :
1 ) Double helical intertwined helix (non channel conformation) found when dissolved in organic
solvents like chloroform or methanol.
2) Single stranded helical dimer or channel form. Why does the channel conformation have amphillic
tryptophan at channel interface?
Figure 0.4:
Channel and Non channel conformations of Gramicidin.
Gramicidin channel formation occurs as seen in Figure (0.4) by two peptides linked head to head
by hydrogen bonds, each forming a half channel. The tryptophan residues along the channel are both
hydrophobic as well as capable of long range electrostatic interactions. The activity of the channel
measured in planar lipid bilayers requires the channel form of the peptide . Gramicidin exhibits a sort
of "memory", based on its storage before addition to the bilayer. If the peptide is stored in methanol
or chloroform the peptides have to establish the pore by rearranging from their non channel form
to channel conformation which is aided by heating or sonication. However, if stored in solvents like
triuroethanol the peptide adopts its channel form instantaneously.
Fully formed Gramicidin channel has an aqueous pore of 4 Åand about 25 Ålong. The pore is
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Figure 0.5:
Left:Gramicidin pore; Right: Monomer arrangement during closed and open conformations.
lined with polar residues and is cation selective. Gating of the pore is seen due to dissociation of
the monomers by breaking of the hydrogen bonds between them. Since the pore is shorter than the
bilayer the pore insertion would cause membrane deformation which is shown in gure (0.5).
Alpha Hemolysin
Alpha Hemolysin is considered an archetype pore for all beta barreled shaped channels. It is a toxin
secreted by Staphylococcus Aureus which causes cell death by perforating the membrane.
Figure 0.6:
Alpha hemolysin : Three parts of the fully formed pore and the water lled transmembrane
channel.
The fully formed pore contains seven subunits which rearrange in the membrane to form a mushroom
shaped pore 100 Åin length and ranging from ≈ 16 - 46 Åin diameter. The peptide sequence of each
monomer reveals a glycine rich hydrophobic region between the C and N terminals. The structure
can be divided into : The cap, The rim and the stem. The cap and the rim contain charged and polar
amino acids where as the bulk of the hydrophobic patch folds into the stem region. The rim houses
7 important aromatic amino acids capable of membrane interactions.
0.1.4 Biomimetic DNA channels
DNA as a building material is based on its unique base pairing abilities. DNA origami technique
exploits this property to "tie up" a long circular DNA, scaold, into a previously conceived structure
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using 200-250 shorter DNA sequences or staples. The shorter DNA sequences are designed to be
complementary to certain parts of the scaold based on which dierent parts are brought together
using Mg to reduce or eliminate the repulsion between negatively charged DNA segments.
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Figure 0.7:
DNA origami technique; a) Depicts a linear single stranded DNA scaold in black. Typically M13
phage of length 7249 base pairs is used. The tiny colored strands represent the staple strands
which are designed to be complementary to specic parts of the scaold using caDNAno software.
b-d) Based on the scaold routing designed as well as staple sequences DNA origami structure is
formed using a one pot reaction technique where all the components are added to a tube and a
heating ramp is applied to allow annealing of the staples to the scaold
Taking inspiration from the biological protein pore structures, DNA origami channels with nanometer
pore dimensions are designed. In order to allow interaction and further incorporation of a negatively
charged hydrophillic structure into the lipid bilayer, the structure is decorated with cholesterol or
tocopherol molecules at its base (much like the "rim" in Alpha Hemolysin). The hydrophobic molecules
are added by using modied staple strands having a 5 or 3 end replaced with the desired molecule.
The versatility of the technique allows to form these channels with dierent shapes and dierent
central pore size. The membrane interaction ability is checked using small unilamellar vesicles which
is a curved lipid bilayer. Transmission electron microscopy is used to indicate interaction tendency of
the modied DNA structures.
The energy required for a channel to penetrate through a membrane would be roughly speaking
the amount of energy required to disrupt the lipid interactions in an area which is the same as that
of the pore. This follows that larger the pore greater will be the energy required to insert it into the
membrane as a larger area of the lipids are cleared away or rearranged to accommodate for the pore.
This energy in DNA channels is provided by favorable interactions of the hydrophobic molecules on
the channel with the lipid tails. Can you calculate the energy required to insert a 4 nm pore into the
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lipid bilayer and how many cholesterols are required to do the same?
Applications of the bioinspired DNA channels are aimed at designing a sensor using resistive pulse
technique (previously introduced) to detect single molecules and distinguish mixtures of molecules via
their electrical signatures. Another exciting eld with potential application for DNA channels is as a
component of an articial cell to allow selective passage of molecules in and out of a compartment.
The ease of modication makes investing in DNA channels lucrative which additionally also sheds
light to basic membrane interaction parameters.
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Figure 0.8:
a)The base of the channel is decorated with hydrophobic molecules which help in providing energy
required for the "stem" of the channel to pierce through the lipid bilayer.b) First two columns
show the various shapes of bio inspired DNA channels that have been realized. The third and
fourth column show the TEM images conrming structure formation and membrane interaction
respectively
0.2 Experimental protocol
0.2.1 Over view
The practical course will provide basic introduction to lipid bilayers and protein channels. The rst
half of the course entails using puried protein pores on painted lipid bilayers. This is done using Orbit
mini setup. The second half of the practical course involves liposome production and imaging. This
part also introduces DNA origami pores, purication methods and nally interaction with liposomes
imaged using uorescence imaging.
0.2.2 Painted bilayer setup and electrical characterization of channels
1) The Orbit mini setup is used for demonstrating electrical activity of ion channels in a lipid bilayer.
The recording is done on a MECA chip containing a 2x2 array of microcavities in a polymer. Each
hole or cavity is associated with an individual integrated Ag/AgCl microelectrode.
2) The chip is inserted to the orbit mini setup and 150 µl of 100 mM KCl is added to wet the chip.
This is indicated by an electrical "open channel" signal.
3) Lipid bilayers are formed on the MECA chip with four channels. DPhPC lipids dissolved in octane
are used at a concentration of 1 mg/ml and painted across the four cavities using a brush. Formation
of the bilayer is observed by reduced or no current passing through the channels otherwise called a
"seal" signal (Indicated by a large resistance in the order of gigaohms.
4) The protein or DNA pores are added to the chip and a V
or a constant voltage is applied to
assist the pore insertion to the bilayer. Once steps of insertions are observed as in (g.1), voltage is
changed to obtain an I-V curve.
hold
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0.2.3 Giant unilamellar vesicles - Inverted emulsion technique
There are several methods for Giant unilamellar vesicle formation. The methodology used here is
called inverted emulsion technique.
1) Lipids dissolved in an organic solvent are processed in a rotary evaporator to produce a uniform
lipid lm under vacuum. This produces a thin lipid lm or cake which is hydrated in subsequent steps
to yeild the GUV formation.
2) The lipid lm is dissolved in mineral oil by repeated heating and vortexing. The solution is sonicated
and allowed to stand at room temperature overnight for maximum dissolution of the lipid into the
mineral oil.
3)The lipid oil mixture is now ready to be processed for further. The 200 µl of the solution is placed
in an eppendorf on ice. 30 µl of the solution designed to be inside the vesicle (usually 200 mM sucrose
solution) is added to the cooled down emulsion and vortexed for 30 s. This step helps in formation
of aqueous droplets surrounded by lipid molecules.The solution is allowed to stand for 30-45 minutes
to stablise the droplets
4) The droplet emulsion is now gently placed on top of the solution intended to be outside the vesicles
(usually 200mM Glucose solution). Care must be taken to adjust the osmolarity of the internal and
external solutions (should be almost equal, outside solution having ≈ 20 mosm more osmolarity helps
in dragging the vesicles to the bottom of the imaging chamber). This allows all the free lipids to line
up at the emulsion external solution interface, creating a layer of free lipids. This is again allowed to
stand for 45 minutes.
5) The column of solution is now gently centrifuged at 12,000 rcf for half hour. This transfers the
lipid surrounded droplets through another layer of the free lipids into the aqueous external solution.
As the droplets move through the interface it zips o a part of the free lipid layer forming a bilayer
along the droplet. This "hole" in the lipid layer at the interface is then lled up by more free lipids
present in the oil solution.
6) The GUVs are present at the bottom of the centrifuge. The oil layer is removed using a pipette
and the vesicles are added to the imaging chamber.
0.2.4 Modication of the DNA pores with Fluorescentlylabeled staples for
imaging
1) DNA origami pores are previously folded in two eppendorfs with a scaold concentration of 50
nM and staple concentration of 150 nM. To one of the tubes 3 fold excess of tocopherol modied
staples (57 possible positions) as well as 3 fold excess of Fluorescentlylabeled staples are added and
incubated for 45 minutes. The second tube is incubated with only Fluorescentlylabeled staples.
2) Both the hydrophobically tagged as well as the plain pores are puried using lter purication.
Millipore lters with a 100 kDa cutos are used for removing the excess staples.The lters are rst
equilibrated with the buer (500 mM KCl) by adding 500 µl of buer to the lter, placing it in the
spin column and centrifuging at 5000 rcf for 5 minutes.The solute is removed from the spin column
after the centrifugation.
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3)450 µl of fresh buer is added to the lter and covered at the top with the sample to be puried.
The lter is centrifuged at 5000 rcf for 5 minutes. The solute is then removed again from the column
and fresh buer is added. The sample is washed with 5 rounds of such centrifugation steps.
4) In order to retrieve the sample the lter is inverted into an empty spin column and spun down at
5000 rcf for 5 minutes. The solution expelled in the spin column now contains the origami sample in
the required buer.
0.2.5 Fluorescence based detection of hydrophobic interactions between DNA
and lipid membranes
1) The imaging chamber is prepared using IBID chambers. 60 µl of the outside solution is added to
the chamber and 5 µl of the GUV solution is added and mixed well. The chamber is kept undisturbed
to allow the GUVs to sink for easier imaging.
2) The GUVs are seen using IX-71 microscope and bright eld images of the vesicles are made.
3) Fluorescentlylabelled DNA pores with and without hydrophobic tags are added to dierent lanes
of the chamber. Colocalization of the pores modied with hydrophobic tags is observed and imaged
in contrast to lack of attachment of the pores to the vesicles in absence of the hydrophobic tags.
0.3 Investigation
The main objective of the practical course is to provide an understanding of how channels and membranes interact. The following questions should be answered after the experimental part is completed.
1) What is the conductance of a Gramicidin pore and Alpha Hemolysin in 100 mM KCl? Illustrate
with trace and steps to deduce.
2) If the energy per lipid due to tail-tail contact is 20 kT, how much energy is required to form a pore
of radius 2.5 nm? Hint : Consider the area in the bilayer reshued due to insertion of the pore.
3) How would the IV curve of a pore change with salt for eg: 100 mM KCl and 100 mM NaCl?
4) Illustrate the inverted emulsion method of vesicle formation diagramatically.
5) What should be the minimum length of an origami channel? Comment on the number of hydrophobic tags requried to allow the pore to insert a bilayer.
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