Lipid Bilayer-Membrane Protein Coupling - Membrane Biophysics, Biological and Medical Physics, Biomedical Engineering - page 97

Table 5.1 Gramicidin sequences and their channel lengths

Gramicidin Abbreviation Sequence Hydrophobic

analogue Channel

Length [ 41 ]Å

[Ala 1 ]gA AgA ( 15 ) f-A-G-A- L -A- V -V- V 22

-W- L -W- L -W- L -W-ea

Des-(D-Val 1 -Gly 2 )gA g A − ( 13 ) f- A -L- A -V- V -V- W 19

-L-W-L-W-L-W-ea

Table 5.2 Phospholipids and their thicknesses
Phospholipid

Abbreviation Bilayer

Thickness
(with n -decane) Å

1,2-dioleoyl- sn -glycero-3-phosphocholine DC 18 : 1 PC 47 . 7 ± 2 . 3[ 16 ]

1,2-diecosenoyl- sn -glycero-3-phosphocholine DC 20 : 1 PC 53 . 9 ± 2 . 5[ 16 ]

1,2-dierucoyl- sn -glycero-3-phosphocholine DC 22 : 1 PC 58 . 4 ± 2 . 5[ 16 ]

1,2-dioleoyl- sn -glycero-3-phosphoethanolamine DOPE Unknown

1,2-dioleoyl- sn -glycero-3-phospho-L-serine DOPS Unknown

Teflon ® separating the two electrolyte solutions of 2.5 mL each, using the pipette
method of Szabo et al. [ 83 ]. All experiments were performed at 25 5 C. The aque-

ous electrolyte solutions were 1.0 M NaCl, buffered to pH 7.0 using 10 mM HEPES
added to the solution. Care was taken to minimize the total amount of lipid (and
n -decane or squalene) that was added; the total volume of the lipid/decane or squa-
lene solution was typically 1,000-times smaller than the volume of the aqueous
solution.
For the experiments with Alm, we added an appropriate amount of Alm from its
10 − 5 M stock solution in dimethyl sulfoxide to the trans side of the lipid bilayer;
the cis side was the electrical ground. In the experiments across DOPE and across
thicker bilayers of DC 20 : 1 PC and DC 22 : 1 PC , we needed a 10-fold and more than
10-100-fold denser solution of Alm compared to that for DC 18 : 1 PC . Experiments
with gA were done in DC 18 : 1 PC / n -decane or squalene, DC 20 : 1 PC /squalene and
DOPE / n -decane bilayers with a 15-residue and a 13-residue gA analog of oppo-
site chirality, e.g. AgA(15) and g A − (13) (added to both sides of the bilayer), an
experimental design that allows for a direct test of how changes in the hydropho-
bic mismatch due to changes in the channel length may affect the channel stabil-
ity. The reason for using gA analogs of opposite chirality is to ensure against the
formation of heterodimers between the 13-amino acid and 15-amino acid analogs
[ 25 , 50 ] which would complicate the data analysis. Using lipids with different acyl
chain lengths results in changing the bilayer thickness, and also in the hydrophobic
mismatch between bilayer thickness and channel lengths (see Tables 5.1 and 5.2 ). In
all experiments with Alm channels, the applied potential across the membrane was
150 mV, and with gA analogs the applied potential was 200 mV.

± 0 .