[95]. Conclusions withdrawn from the electrochemical and in situ PM IRRAS
experiments indicate a potential-driven formation of alamethicin ion-conductive
channels in the lipid bilayer. The orientation of the AMP depends on the potential,
while the orientation of the DPhPC lipids in the membrane is unaffected by the
potentials applied to the electrode. These results conclude that alamethicin interacts
with membrane via barrel-stave mechanism (see Fig. 3.15b, IV) [95, 98]. The
availability of the molecular-scale picture of potential-driven changes induced by
the interaction of alamethicin with model lipid membranes allowed an investigation
of the impact of a drug, blocking the ion transport through the membrane, on
potential-driven changes in a DMPC:EggPG:alamethicin (9:9:2 molar ratio) bilayer
[94]. Amiloride and its derivatives are commercially available drugs. In the presence
of 60 μM amiloride in the electrolyte solution the PM IRRA spectra originating from
the lipid molecules undergo substantial changes compared to the behavior observed
in the absence of the drug. The average tilt angle of the hydrocarbon chains in lipid
molecules is close to 45
versus surface normal [94]. In the absence of the drug in the
bilayer adsorbed on the Au surface the hydrocarbon chains have an average tilt of
37
–40
. In the presence of amiloride in the electrolyte solution the tilt of the
hydrocarbon chains in lipid molecules increases indicating that the hydrocarbon
chains are more disordered. The analysis of in situ PM IRRA spectra in the amide I
0
region of alamethicin displays no differences in the potential-dependent peptide
orientation in the absence and presence of amiloride. These results show that
amiloride has a distortive effect on the membrane lipids, but it has no effect on the
insertion of alamethicin into the membrane. Described above examples illustrate that
PM IRRAS with electrochemical control is applicable for simultaneous studies of
the potential dependent changes in the conformation and orientation of both lipid and
polypeptide components in the supramolecular assembly of the model cell
membrane.
Interaction of Lipid Bilayers with Proteins
Proteins have a more complex structure than AMPs. The presence of different
secondary structure elements is reflected in the amide I mode of the IR spectra of
proteins. In other words, the amide I mode of proteins is broad and composed of
several overlapped IR absorption bands. Due to the structural complexity the
deconvolution of the amide I
0 mode of proteins is more difficult than of AMPs.
Examples of the application of in situ PM IRRAS for studies of structural changes in
the lipid membrane interacting specifically with an anchor (recoverin) and saccharide binding (siglec-4 and cholera toxin) proteins are described below.
Recoverin belongs to Ca
2+ -dependent proteins which contain a covalently
attached myristoyl chain at the N-terminus. This flexible chain anchors the protein
into the hydrophobic part of fluid lipid membranes [102–106]. The biochemical
properties of recoverin indicate that the insertion of the protein myristoyl chain into
the membrane leads to changes in the conformation and/or orientation of the protein.
The interaction of native myristoylated (Rv) and wild-type non-myristoylated (n-Rv)
recoverin with model membranes was investigated using in situ PM IRRAS [39, 44,
45]. The DMPC:cholesterol (7:3 molar ratio) bilayer was LB-LS transferred on the
76
3 In Situ PM IRRAS Studies of Redox-Inactive Molecular Films Adsorbed on. . .
experiments indicate a potential-driven formation of alamethicin ion-conductive
channels in the lipid bilayer. The orientation of the AMP depends on the potential,
while the orientation of the DPhPC lipids in the membrane is unaffected by the
potentials applied to the electrode. These results conclude that alamethicin interacts
with membrane via barrel-stave mechanism (see Fig. 3.15b, IV) [95, 98]. The
availability of the molecular-scale picture of potential-driven changes induced by
the interaction of alamethicin with model lipid membranes allowed an investigation
of the impact of a drug, blocking the ion transport through the membrane, on
potential-driven changes in a DMPC:EggPG:alamethicin (9:9:2 molar ratio) bilayer
[94]. Amiloride and its derivatives are commercially available drugs. In the presence
of 60 μM amiloride in the electrolyte solution the PM IRRA spectra originating from
the lipid molecules undergo substantial changes compared to the behavior observed
in the absence of the drug. The average tilt angle of the hydrocarbon chains in lipid
molecules is close to 45
versus surface normal [94]. In the absence of the drug in the
bilayer adsorbed on the Au surface the hydrocarbon chains have an average tilt of
37
–40
. In the presence of amiloride in the electrolyte solution the tilt of the
hydrocarbon chains in lipid molecules increases indicating that the hydrocarbon
chains are more disordered. The analysis of in situ PM IRRA spectra in the amide I
0
region of alamethicin displays no differences in the potential-dependent peptide
orientation in the absence and presence of amiloride. These results show that
amiloride has a distortive effect on the membrane lipids, but it has no effect on the
insertion of alamethicin into the membrane. Described above examples illustrate that
PM IRRAS with electrochemical control is applicable for simultaneous studies of
the potential dependent changes in the conformation and orientation of both lipid and
polypeptide components in the supramolecular assembly of the model cell
membrane.
Interaction of Lipid Bilayers with Proteins
Proteins have a more complex structure than AMPs. The presence of different
secondary structure elements is reflected in the amide I mode of the IR spectra of
proteins. In other words, the amide I mode of proteins is broad and composed of
several overlapped IR absorption bands. Due to the structural complexity the
deconvolution of the amide I
0 mode of proteins is more difficult than of AMPs.
Examples of the application of in situ PM IRRAS for studies of structural changes in
the lipid membrane interacting specifically with an anchor (recoverin) and saccharide binding (siglec-4 and cholera toxin) proteins are described below.
Recoverin belongs to Ca
2+ -dependent proteins which contain a covalently
attached myristoyl chain at the N-terminus. This flexible chain anchors the protein
into the hydrophobic part of fluid lipid membranes [102–106]. The biochemical
properties of recoverin indicate that the insertion of the protein myristoyl chain into
the membrane leads to changes in the conformation and/or orientation of the protein.
The interaction of native myristoylated (Rv) and wild-type non-myristoylated (n-Rv)
recoverin with model membranes was investigated using in situ PM IRRAS [39, 44,
45]. The DMPC:cholesterol (7:3 molar ratio) bilayer was LB-LS transferred on the
76
3 In Situ PM IRRAS Studies of Redox-Inactive Molecular Films Adsorbed on. . .