Biomedical Engineering Reference
In-Depth Information
1. ReconstitutionofOmpF
In Section II, the electrochemical characteristics of pore-spanning
membranes investigated by EIS were discussed in detail. The data
reveal that the absolute value of the impedance |
Z
| at frequencies
< 10
1
Hz is solely governed by the resistive properties of the bi-
layer and is thus frequency independent. Thereby, performing EIS
experiments at a fixed frequency of 10 Hz or less allows following
the membrane resistance
R
m
without taking the capacitive proper-
ties into account. Membrane-integrated, active ion channels pro-
duce a change in membrane conductivity, which is readily detected
as a change in the absolute value of the impedance |
Z
| at the chosen
frequency as a function of time. Prior to this time resolved experi-
ment, impedance spectra need to be recorded to determine the fre-
quency range in which the membrane resistance
R
m
is well de-
fined.
As mentioned in Section III, reconstitution of transmembrane
proteins from an aqueous solution requires that the molecules are
solubilised in and added from a detergent solution. Detergents are
amphiphiles and known to interact with lipid bilayers. Studies on
liposomes revealed that detergents at concentrations considerably
below their critical micellar concentration (CMC) partition into the
bilayers and permeabilise them.
72,73
Hence, prior to protein addi-
tion, we first investigated the influence of the detergent
o
-POE on
time monitored in 0.1 M NaCl. It is obvious that a final concentra-
tion of 0.0002% (
v
/
v
)
o
-POE, which is four orders of magnitude
below its CMC, is sufficient to permeabilise the bilayer such that
|
Z
|(1 Hz) decreases by two orders of magnitude. However, by ex-
changing for detergent-free electrolyte solution, the original mem-
brane resistance is recovered. Thus, the interaction of
o
-POE in
sub-CMC concentrations with pore-spanning membranes is fully
reversible. Then, OmpF in
o
-POE (0.0002% (
v
/
v
)
o
-POE, 0.5 nM
OmpF) was added to the pore-spanning lipid bilayers, again result-
ing in a significant decrease in membrane resistance. Yet, rinsing
with electrolyte solution resulted in only partial recovery of the
membrane resistance. The net drop in bilayer resistance
R
m
is a
result of the formation of OmpF pores in the pore-suspending
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