n-Rv is ascribed to the existence of the protein in so called tense-state. This state has
been found in Ca
2+ -free bound Rv with myristoyl chain buried into the protein and in
n-Rv [107]. In the presence of Ca
2+ ions the myristoyl chain in Rv is extruded,
transforming Rv into a relaxed state. The conformation of the anchor protein is
different in the membrane-bound and dissociated states.
Moreover, the deconvolution of the amide I
0 mode of the membrane bound Rv
and n-Rv shows that the content of α-helical structural elements depends on the
membrane potential [45]. It is lower at positive transmembrane potentials and
increases with a negative potential shift. These changes are fully reversible with
respect to the potential applied to the Au electrode in the successive scans. Changes
in the content of the α-helices in recoverins may indicate:
1. Denaturation of the protein;
2. Reorientation of the protein as a function of the membrane potential.
Denaturation of a protein usually leads to some irreversible changes in the amide I
mode spectral region. Spectral changes in the amide I
0 mode region reported by
Brand [45] are reversible as a function of the electrode potential. In Rv dissolved in
the electrolyte solution the content of the α-helices is equal to 52% and in n-Rv—
55%. Considering these statements, the following conclusions are proposed:
1. Changes in the content of the α-helical structural elements in membrane bound
Rv and n-Rv as a function of the membrane potential are due to conformation
changes and reorientations of the membrane bound protein;
2. During the potential scan no denaturation of the protein is observed;
3. At random distribution of the protein in the membrane the expected content of the
α-helical fragments is close to 50–55% [45, 104, 108].
Taking this into account, the order parameter of the amide I
0 mode assigned to
α-helices and the order parameter of α-helices in the proteins were calculated
(Eqs. 3.1–3.3) [45]. From the order parameter of α-helices the average orientation
of 11 α-helical fragments in recoverin is calculated as shown in Fig. 3.22.
Figure 3.22 shows that both Rv and n-Rv have the same orientation of their
α-helical fragments in the membrane bound state. The orientation of the adsorbed
protein changes as a function of the transmembrane potential. In recoverin the
α-helical fragments have well defined arrangement with respect to the orientation
of the extruded myristoyl chain. Figure 3.23a illustrates the structure of recoverin.
The structure of recoverin, shown in Fig. 3.23a, corresponds to the orientation of
the protein bound to a lipid monolayer at the air|water interface [109]. The α-helices
in recoverin have a preferential parallel to the plane of the lipid monolayer plane
orientation. At positive transmembrane potentials, in the membrane bound state, the
average tilt of 11 α-helical fragments in Rv and n-Rv is close to 65
–55
(Fig. 3.22).
The α-helices have also a preferential parallel to the bilayer plane orientation
(Fig. 3.23b). Thus, the arrangement of recoverin interacting with the lipid bilayer
is similar to that found in the monolayer assembly at the air|water interface. A
negative shift of the transmembrane potential leads to a gradual decrease in the
average tilt of the long axis in α-helical fragments of recoverins (Fig. 3.22). The
3.3 In Situ PM IRRAS Studies of Films of Biomolecules Adsorbed on Electrode Surfaces 81
been found in Ca
2+ -free bound Rv with myristoyl chain buried into the protein and in
n-Rv [107]. In the presence of Ca
2+ ions the myristoyl chain in Rv is extruded,
transforming Rv into a relaxed state. The conformation of the anchor protein is
different in the membrane-bound and dissociated states.
Moreover, the deconvolution of the amide I
0 mode of the membrane bound Rv
and n-Rv shows that the content of α-helical structural elements depends on the
membrane potential [45]. It is lower at positive transmembrane potentials and
increases with a negative potential shift. These changes are fully reversible with
respect to the potential applied to the Au electrode in the successive scans. Changes
in the content of the α-helices in recoverins may indicate:
1. Denaturation of the protein;
2. Reorientation of the protein as a function of the membrane potential.
Denaturation of a protein usually leads to some irreversible changes in the amide I
mode spectral region. Spectral changes in the amide I
0 mode region reported by
Brand [45] are reversible as a function of the electrode potential. In Rv dissolved in
the electrolyte solution the content of the α-helices is equal to 52% and in n-Rv—
55%. Considering these statements, the following conclusions are proposed:
1. Changes in the content of the α-helical structural elements in membrane bound
Rv and n-Rv as a function of the membrane potential are due to conformation
changes and reorientations of the membrane bound protein;
2. During the potential scan no denaturation of the protein is observed;
3. At random distribution of the protein in the membrane the expected content of the
α-helical fragments is close to 50–55% [45, 104, 108].
Taking this into account, the order parameter of the amide I
0 mode assigned to
α-helices and the order parameter of α-helices in the proteins were calculated
(Eqs. 3.1–3.3) [45]. From the order parameter of α-helices the average orientation
of 11 α-helical fragments in recoverin is calculated as shown in Fig. 3.22.
Figure 3.22 shows that both Rv and n-Rv have the same orientation of their
α-helical fragments in the membrane bound state. The orientation of the adsorbed
protein changes as a function of the transmembrane potential. In recoverin the
α-helical fragments have well defined arrangement with respect to the orientation
of the extruded myristoyl chain. Figure 3.23a illustrates the structure of recoverin.
The structure of recoverin, shown in Fig. 3.23a, corresponds to the orientation of
the protein bound to a lipid monolayer at the air|water interface [109]. The α-helices
in recoverin have a preferential parallel to the plane of the lipid monolayer plane
orientation. At positive transmembrane potentials, in the membrane bound state, the
average tilt of 11 α-helical fragments in Rv and n-Rv is close to 65
–55
(Fig. 3.22).
The α-helices have also a preferential parallel to the bilayer plane orientation
(Fig. 3.23b). Thus, the arrangement of recoverin interacting with the lipid bilayer
is similar to that found in the monolayer assembly at the air|water interface. A
negative shift of the transmembrane potential leads to a gradual decrease in the
average tilt of the long axis in α-helical fragments of recoverins (Fig. 3.22). The
3.3 In Situ PM IRRAS Studies of Films of Biomolecules Adsorbed on Electrode Surfaces 81