the conformation, packing, and orientation of both, lipid and peptides molecules.
This approach facilitates distinction between different modes of action of AMPs. In
situ PM IRRAS was used to investigate the interaction of model cell membranes
with gramicidin [41], bacteriocin-like peptide BacSp222 [43], and alamethicin [94–
96]. The mechanism of interaction of alamethicin was studied on floating [95],
tethered [95] and supported on the Au surface [96] lipid bilayers. Alamethicin is
an AMP composed of 20 amino acids. It has a helical structure with an α-helix at the
N-terminus and 3 10 -helix at the C-terminus [97]. Lipid bilayers containing 1,2-di-Ophytanyl-sn-glycero3-phosphocholine (DPhPC) and alamethicin (9:1 mole ratio) in
each leaflet were transferred onto a bare and thioglucose modified Au electrode
surface. Electrochemical and spectroscopic characteristics of both model bilayers are
similar. In the potential range À0.1 > E > 0.0 V versus Ag/AgCl the capacitance of
the DPhPC:alamethicin bilayer reaches a minimum (8 μF cm
À2 ) [95, 98]. At
E < À0.1 V an increase in the membrane capacitance and decrease in the membrane
resistance are observed. In situ PM IRRAS was done to examine structural changes
in the lipid bilayer during interactions with alamethicin. The analysis of the CH
stretching modes region of the supported and floating DPhPC:alamethicin bilayers
provides information on the packing of the hydrophobic hydrocarbon chain fragments in the membrane. The maxima of absorption of the methylene stretching
modes indicate that the hydrocarbon chains in DPhPC molecules exist in a liquid
state. Despite the liquid state of the hydrocarbon chains, the analysis of the intensities of the methylene stretching modes indicates the presence of long range order in
the hydrophobic fragment of the bilayer. The integral intensities of the methylene
stretching modes are independent of the potential applied to the Au electrode. The
average tilt of the hydrocarbon chains in the supported and floating DPhPC:
alamethicin bilayers is in the rage of 30
–35
versus surface normal. PM IRRA
spectra in the 1800–1600 cm
À1 region contain two IR absorption modes: the
ν(C¼O) in the carbonyl ester group in DPhPC and the amide I mode in alamethicin.
The PM IRRA spectra of the floating DPhPC:alamethicin bilayer are shown in
Fig. 3.16.
The top curve in Fig. 3.16 shows the calculated PM IRRA spectrum of randomly
distributed DPhPC and alamethicin molecules in a bilayer thick film. In this spectrum the ν(C¼O) mode of the carbonyl ester stretching mode in the DPhDP lipid
(1750–1720 cm
À1 ) is strong. In contrast, in the PM IRRA spectra of the floating
DPhPC:alamethicin bilayer the ν(C¼O) mode is very weak (Fig. 3.16). The attenuation of the ν(C¼O) mode in the floating bilayer indicates that the ester carbonyl
groups in the lipid molecules make a large angle with respect to the surface normal.
The orientation of the C¼O group in the bilayer does not change with potential. The
broad IR absorption mode in the 1700–1600 cm
À1 spectral region arises from the
amide I mode in alamethicin. The deconvolution of the overall amide I
0 mode
(measured in D 2 O) gives five modes centered at 1620, 1633, 1646, 1656 and
1673 cm
À1 [98]. In the membrane associated alamethicin two amide I
0 modes
(at 1633 and 1656 cm
À1 ) are strong. They arise from the 3 10 -helix and α-helix
secondary structure elements, respectively (Table 3.3). The very weak mode at
1620 cm
À1 is ascribed to β-sheet, while two other modes at 1646 and 1673 cm
À1
3.3 In Situ PM IRRAS Studies of Films of Biomolecules Adsorbed on Electrode Surfaces 73
This approach facilitates distinction between different modes of action of AMPs. In
situ PM IRRAS was used to investigate the interaction of model cell membranes
with gramicidin [41], bacteriocin-like peptide BacSp222 [43], and alamethicin [94–
96]. The mechanism of interaction of alamethicin was studied on floating [95],
tethered [95] and supported on the Au surface [96] lipid bilayers. Alamethicin is
an AMP composed of 20 amino acids. It has a helical structure with an α-helix at the
N-terminus and 3 10 -helix at the C-terminus [97]. Lipid bilayers containing 1,2-di-Ophytanyl-sn-glycero3-phosphocholine (DPhPC) and alamethicin (9:1 mole ratio) in
each leaflet were transferred onto a bare and thioglucose modified Au electrode
surface. Electrochemical and spectroscopic characteristics of both model bilayers are
similar. In the potential range À0.1 > E > 0.0 V versus Ag/AgCl the capacitance of
the DPhPC:alamethicin bilayer reaches a minimum (8 μF cm
À2 ) [95, 98]. At
E < À0.1 V an increase in the membrane capacitance and decrease in the membrane
resistance are observed. In situ PM IRRAS was done to examine structural changes
in the lipid bilayer during interactions with alamethicin. The analysis of the CH
stretching modes region of the supported and floating DPhPC:alamethicin bilayers
provides information on the packing of the hydrophobic hydrocarbon chain fragments in the membrane. The maxima of absorption of the methylene stretching
modes indicate that the hydrocarbon chains in DPhPC molecules exist in a liquid
state. Despite the liquid state of the hydrocarbon chains, the analysis of the intensities of the methylene stretching modes indicates the presence of long range order in
the hydrophobic fragment of the bilayer. The integral intensities of the methylene
stretching modes are independent of the potential applied to the Au electrode. The
average tilt of the hydrocarbon chains in the supported and floating DPhPC:
alamethicin bilayers is in the rage of 30
–35
versus surface normal. PM IRRA
spectra in the 1800–1600 cm
À1 region contain two IR absorption modes: the
ν(C¼O) in the carbonyl ester group in DPhPC and the amide I mode in alamethicin.
The PM IRRA spectra of the floating DPhPC:alamethicin bilayer are shown in
Fig. 3.16.
The top curve in Fig. 3.16 shows the calculated PM IRRA spectrum of randomly
distributed DPhPC and alamethicin molecules in a bilayer thick film. In this spectrum the ν(C¼O) mode of the carbonyl ester stretching mode in the DPhDP lipid
(1750–1720 cm
À1 ) is strong. In contrast, in the PM IRRA spectra of the floating
DPhPC:alamethicin bilayer the ν(C¼O) mode is very weak (Fig. 3.16). The attenuation of the ν(C¼O) mode in the floating bilayer indicates that the ester carbonyl
groups in the lipid molecules make a large angle with respect to the surface normal.
The orientation of the C¼O group in the bilayer does not change with potential. The
broad IR absorption mode in the 1700–1600 cm
À1 spectral region arises from the
amide I mode in alamethicin. The deconvolution of the overall amide I
0 mode
(measured in D 2 O) gives five modes centered at 1620, 1633, 1646, 1656 and
1673 cm
À1 [98]. In the membrane associated alamethicin two amide I
0 modes
(at 1633 and 1656 cm
À1 ) are strong. They arise from the 3 10 -helix and α-helix
secondary structure elements, respectively (Table 3.3). The very weak mode at
1620 cm
À1 is ascribed to β-sheet, while two other modes at 1646 and 1673 cm
À1
3.3 In Situ PM IRRAS Studies of Films of Biomolecules Adsorbed on Electrode Surfaces 73