Lipid Bilayer-Membrane Protein Coupling - Membrane Biophysics, Biological and Medical Physics, Biomedical Engineering - page 96

5.4 Experimental Studies of the Functions of Gramicidin A
and Alamethicin Channels in Lipid Membranes

So far we have discussed theoretical aspects of both gramicidin A and alame-
thicin channel functions in lipid membranes. We have successfully addressed how
membrane protein functions become regulated due to energetic lipid bilayer mem-
brane protein coupling, using a very traditional theoretical approach which involves
screened Coulomb interactions [ 9 ]. Although both of these channels have been exten-
sively experimentally investigated, we reiterate some of the aspects of the experi-
mental studies using our own investigations (see [ 11 ] in Chap. 4 ) . The rather novel
parameters emerging from the studies help to validate the theoretical approaches
explained in earlier sections. These experimental studies were done by Md Ashra-
fuzzaman in collaboration with Dr. Olaf Sparre Andersen during his tenure in Cornell
University Weill Medical College.

5.4.1 Materials and Methods

Materials

1,2-Dioleoyl-sn-Glycero-3-Phosphocholine ( DC 18 : 1 PC ), 1,2-Diecosenoyl-sn-
Glycero-3-Phosphocholine ( DC 20 : 1 PC ), 1,2-Dierucoyl-sn-Glycero-3-Phosphocho-
line ( DC 22 : 1 PC ), 1,2-dioleoyl-sn-glycero-3-phosphoethanol-amine ( DOPE ), and
1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] ( DOPS ) were from Avanti Polar
Lipid (Alabaster, AL, USA) and used without further purification. n -Decane was
99.9 % pure from ChemSampCo (Trenton, NJ, USA) and squalene (squalene was
filtered through chromatographic alumina (acid type) from Sigma to make it radical-
free) was from Sigma (St. Louis, MO, USA). Alamethicin (Alm), an antibiotic from
Tricoderma viride that is a mixture of alamethicin homologs, was from Sigma. Gram-
icidin A (gA) analogue

Ala 1 gA (with 15 amino acids in the sequence) (AgA(15))

and the sequence-shortened analogue, des-(D

[ ]

Va l 1 Gly 2 )-gA (with 13 amino − −

acids in the sequence) ( g A − (

) were generous gifts from Drs. R.E. Koeppe II
and D.V. Greathouse (see [11] in Chap. 4 ) . They were synthesized and purified as
described in [ 31 ]. The amino acid sequences, channel lengths, phospholipids, bilayer
thicknesses and abbreviations used in this article are shown in Tables 5.1 and 5.2 .
The electrolyte solution (NaCl) was buffered with N-2-Hydroxyethylpiperazine-N'-
2-ethanesulfonic Acid (HEPES) (pH 7.0) and was from Sigma.

13 )

Methods

Planar lipid bilayers were formed from DC 18 : 1 PC , DC 20 : 1 PC , DC 22 : 1 PC , DOPE
or DOPS / n -decane or squalene (2.5 % w/v) solutions across a 1.5 mm hole in