222
B. Czarnik-Matusewicz and Y.M. Jung
RNase A has also indicated that structural rearrangement most likely initiated the
aggregation process, as was observed for hemoglobin [63]. 2dCoS studies of the
aggregation of hemoglobin and RNase A did not confirm the then surprising results
obtained for myoglobin [61].
the aggregation process induced by temperature and by ph has been studied
in whole porcine plasma (serum albumin + globulins + fibrinogen), serum (serum
albumin + globulins), and its fractions (serum, albumin and globulins) [82, 88]. Infrared results aided by 2dCoS have shown that the behavior of serum albumin and
globulins during the thermal treatment has been strongly changed in the presence
of fibrinogen. despite the fibrinogen-correlated differences, in all the systems, the
α-helical structure has been the most temperature-sensitive conformation. The disappearance of the α-helical structure and formation of the heat-induced intermolecular β-sheet structure proceeded in a closely related way.
temperature could be a very advantageous perturbation in studies of the interactions between intrinsic and extrinsic proteins and lipid bilayers because during
the heating process, lipid bilayers undergo a characteristic phase transition from
a gel to a fluid state; this transition could act as an additional internal perturbation of the protein structure. Such 2dCoS-aided investigations were performed for
the first time by Lefèvre and Subirade [54] who studied the interactions between
β-lactoglobulin (β-lg) and dimyristoylphosphatidylglycerol (DMPG) bilayers at pD
above (pd 7.4) and below (pd 4.4) the isoelectric point of the protein (pI = 5.2). the
spectra were measured for β-lg alone and in the presence of DMPG as a function
of temperature over the range of the main transition of dmPg to determine whether the conformational modifications of the protein in the presence of lipids were
caused by thermal effects or whether the conformations were modulated by the
chain-melting phase transition. to answer this question, synchronous and asynchronous 2d correlation maps were constructed from the spectral variations in the two
frequency ranges, i.e., 3,000–2,800 cm
−1
and 1,685–1,600 cm
−1
. the heat-induced
structural changes of β-lg did not correlate with the conformational modifications
of the phospholipids along the phase transition. the thermal behavior of the protein
was not modulated by the lipid chain melting; instead, the behavior represented the
heat-induced protein rearrangement in the presence of dmPg. moreover, analysis
of the sequence of events during the thermal unfolding of β-lg at pD 4.4 and 7.4
has shown similar initial structures and similar subsequent thermal effects under the
conditions of the two pd values.
the influence of temperature and time on the denaturation and aggregation phenomena of cytochrome c (cyt c) in the presence of dmPg was investigated in [56].
Aggregation at 65 °C has been confirmed by a band at 1,616 cm
−1
, which is an IR
signature of this process. the studies assisted by 2dCoS have revealed that aggregation starts between nearly native proteins whose unfolding leads to further
aggregation of cyt c. In a later stage of this process, the formation of intermolecular
bonds and the unfolding of the α-helices appear to be simultaneous. Based on this
finding, a two-step aggregation process was proposed. 2dCoS results obtained from
data collected during the heating period have shown a loosening of the tertiary
B. Czarnik-Matusewicz and Y.M. Jung
RNase A has also indicated that structural rearrangement most likely initiated the
aggregation process, as was observed for hemoglobin [63]. 2dCoS studies of the
aggregation of hemoglobin and RNase A did not confirm the then surprising results
obtained for myoglobin [61].
the aggregation process induced by temperature and by ph has been studied
in whole porcine plasma (serum albumin + globulins + fibrinogen), serum (serum
albumin + globulins), and its fractions (serum, albumin and globulins) [82, 88]. Infrared results aided by 2dCoS have shown that the behavior of serum albumin and
globulins during the thermal treatment has been strongly changed in the presence
of fibrinogen. despite the fibrinogen-correlated differences, in all the systems, the
α-helical structure has been the most temperature-sensitive conformation. The disappearance of the α-helical structure and formation of the heat-induced intermolecular β-sheet structure proceeded in a closely related way.
temperature could be a very advantageous perturbation in studies of the interactions between intrinsic and extrinsic proteins and lipid bilayers because during
the heating process, lipid bilayers undergo a characteristic phase transition from
a gel to a fluid state; this transition could act as an additional internal perturbation of the protein structure. Such 2dCoS-aided investigations were performed for
the first time by Lefèvre and Subirade [54] who studied the interactions between
β-lactoglobulin (β-lg) and dimyristoylphosphatidylglycerol (DMPG) bilayers at pD
above (pd 7.4) and below (pd 4.4) the isoelectric point of the protein (pI = 5.2). the
spectra were measured for β-lg alone and in the presence of DMPG as a function
of temperature over the range of the main transition of dmPg to determine whether the conformational modifications of the protein in the presence of lipids were
caused by thermal effects or whether the conformations were modulated by the
chain-melting phase transition. to answer this question, synchronous and asynchronous 2d correlation maps were constructed from the spectral variations in the two
frequency ranges, i.e., 3,000–2,800 cm
−1
and 1,685–1,600 cm
−1
. the heat-induced
structural changes of β-lg did not correlate with the conformational modifications
of the phospholipids along the phase transition. the thermal behavior of the protein
was not modulated by the lipid chain melting; instead, the behavior represented the
heat-induced protein rearrangement in the presence of dmPg. moreover, analysis
of the sequence of events during the thermal unfolding of β-lg at pD 4.4 and 7.4
has shown similar initial structures and similar subsequent thermal effects under the
conditions of the two pd values.
the influence of temperature and time on the denaturation and aggregation phenomena of cytochrome c (cyt c) in the presence of dmPg was investigated in [56].
Aggregation at 65 °C has been confirmed by a band at 1,616 cm
−1
, which is an IR
signature of this process. the studies assisted by 2dCoS have revealed that aggregation starts between nearly native proteins whose unfolding leads to further
aggregation of cyt c. In a later stage of this process, the formation of intermolecular
bonds and the unfolding of the α-helices appear to be simultaneous. Based on this
finding, a two-step aggregation process was proposed. 2dCoS results obtained from
data collected during the heating period have shown a loosening of the tertiary