Positions of the absorption maxima of the CH 2 stretching modes
[ν as (CH 2 ) at 2923 Æ 2 cm
À1 and (ν s (CH 2 ) at 2951 Æ 2 cm
À1 ] indicate that
the hydrocarbon chains exist in a gel phase. Neither the electric potentials nor the
presence of siglec-4 in the electrolyte solution affect the integral intensities of the
methylene stretching modes (Fig. 3.24). The methylene stretching modes are
strongly attenuated, indicating an almost vertical to the bilayer plane orientation of
the hydrocarbon chains in lipid molecules in the studied bilayer. Binding of siglec-4
protein to the gangliosides is confirmed by the presence of strong, broad amide I
0
mode in the PM IRRA spectra [39]. The protein-carbohydrate interaction is
expressed by the appearance of a new amide I
0 absorption mode at 1617 cm
À1 .
This result indicates changes in the β-sheet structural elements of siglec-4 in the
protein bound to gangliosides on the membrane surface. In the desorbed membrane
this mode disappears from the PM IRRA spectra, indicating dissociation of the weak
protein-carbohydrate binding.
Cholera toxin binds specifically to the GM 1 ganglioside present on the membrane
surface [42]. Binding of cholera toxin to the lipid bilayer, similarly to the binding of
siglec-4, has little effect on the orientation of the lipid molecules in the membrane.
Figure 3.25 shows the PM IRRA spectra in the amide I
0 mode region of cholera toxin
bound to GM1 in the outer leaflet of an asymmetric membrane. The amide I
0 mode of
cholera toxin is composed of several overlapped IR absorption modes.
Deconvolution of this mode gives seven IR absorption modes centered at 1618,
1627, 1642, 1655, 1667, 1673 and 1691 cm
À1 (see Fig. 3.25b), This spectrum
reflects the complex structure of cholera toxin [42].
A weak IR absorption mode at 1618 cm
À1 is ascribed to the side chains vibration.
The amide I
0 modes at 1627 and 1673 cm
À1 arise from the antiparallel β-sheet and
α-helix structural elements, respectively. Two other modes centered at 1642 and
1667 cm
À1 are assigned to random structures (Fig. 3.25b). In cholera toxin the
α-helices are located in the inner channel of the five subunits forming the protein
[117]. Their average tilt versus the symmetry axis of the pentamer (axis of the pore,
major axis) protein is equal to 18.7
versus surface normal [42]. Integral intensities
of the amide I
0 modes arising from α-helices and β-sheet structural elements were
analyzed as a function of potential applied to the Au electrode (transmembrane
potential). Figure 3.26 shows the plot of the tilt angle of the major axis of the
α-helices in cholera toxin as the function of the potential applied to the lipid bilayer
assembled on the Au electrode surface.
At positive potentials the average tilt angle of the long axes of α-helices is close to
20
(Fig. 3.26). These results indicate that the channel is open and the α-helices
orient themselves almost normal to the membrane surface. At negative potentials an
increase in the tilt angle of the α-helix axes to ca. 40
is observed (Fig. 3.26).
3.3 In Situ PM IRRAS Studies of Films of Biomolecules Adsorbed on Electrode Surfaces 85
[ν as (CH 2 ) at 2923 Æ 2 cm
À1 and (ν s (CH 2 ) at 2951 Æ 2 cm
À1 ] indicate that
the hydrocarbon chains exist in a gel phase. Neither the electric potentials nor the
presence of siglec-4 in the electrolyte solution affect the integral intensities of the
methylene stretching modes (Fig. 3.24). The methylene stretching modes are
strongly attenuated, indicating an almost vertical to the bilayer plane orientation of
the hydrocarbon chains in lipid molecules in the studied bilayer. Binding of siglec-4
protein to the gangliosides is confirmed by the presence of strong, broad amide I
0
mode in the PM IRRA spectra [39]. The protein-carbohydrate interaction is
expressed by the appearance of a new amide I
0 absorption mode at 1617 cm
À1 .
This result indicates changes in the β-sheet structural elements of siglec-4 in the
protein bound to gangliosides on the membrane surface. In the desorbed membrane
this mode disappears from the PM IRRA spectra, indicating dissociation of the weak
protein-carbohydrate binding.
Cholera toxin binds specifically to the GM 1 ganglioside present on the membrane
surface [42]. Binding of cholera toxin to the lipid bilayer, similarly to the binding of
siglec-4, has little effect on the orientation of the lipid molecules in the membrane.
Figure 3.25 shows the PM IRRA spectra in the amide I
0 mode region of cholera toxin
bound to GM1 in the outer leaflet of an asymmetric membrane. The amide I
0 mode of
cholera toxin is composed of several overlapped IR absorption modes.
Deconvolution of this mode gives seven IR absorption modes centered at 1618,
1627, 1642, 1655, 1667, 1673 and 1691 cm
À1 (see Fig. 3.25b), This spectrum
reflects the complex structure of cholera toxin [42].
A weak IR absorption mode at 1618 cm
À1 is ascribed to the side chains vibration.
The amide I
0 modes at 1627 and 1673 cm
À1 arise from the antiparallel β-sheet and
α-helix structural elements, respectively. Two other modes centered at 1642 and
1667 cm
À1 are assigned to random structures (Fig. 3.25b). In cholera toxin the
α-helices are located in the inner channel of the five subunits forming the protein
[117]. Their average tilt versus the symmetry axis of the pentamer (axis of the pore,
major axis) protein is equal to 18.7
versus surface normal [42]. Integral intensities
of the amide I
0 modes arising from α-helices and β-sheet structural elements were
analyzed as a function of potential applied to the Au electrode (transmembrane
potential). Figure 3.26 shows the plot of the tilt angle of the major axis of the
α-helices in cholera toxin as the function of the potential applied to the lipid bilayer
assembled on the Au electrode surface.
At positive potentials the average tilt angle of the long axes of α-helices is close to
20
(Fig. 3.26). These results indicate that the channel is open and the α-helices
orient themselves almost normal to the membrane surface. At negative potentials an
increase in the tilt angle of the α-helix axes to ca. 40
is observed (Fig. 3.26).
3.3 In Situ PM IRRAS Studies of Films of Biomolecules Adsorbed on Electrode Surfaces 85