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8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
structure, which preceded the aggregation process. Additionally, an increased number of β-turns at the expense of β-sheet content was reported. That study confirmed
the potential of 2d-IR-CoS spectroscopy to investigate the intrinsic temperaturemediated or direct lipid-mediated change in protein conformation in a lipid bilayer
environment.
It is outside of the scope of this article to mention all of the results in which
temperature perturbation has been applied in 2d infrared correlation spectroscopy.
the selected examples are intended to illustrate that there is only one step between
the very common near-IR/mid-IR temperature-dependent experiment and 2d correlation spectroscopy. Why should this step be taken? A clear answer arises from the
above examples: In contrast to ordinary one-dimensional analysis, two-dimensional
infrared correlation spectroscopy can provide insight at the molecular level into the
underlying mechanisms responsible for protein unfolding, including protein aggregation and fibrillation. Can we expect anything more from a routine temperaturedependent IR experiment?
8.3.3 Concentration of protein
Concentration has been a commonly used perturbation in investigations by ozaki
and co-workers. variations in the concentration of protein at a given temperature
have been the main source of spectral changes subjected to 2dCoS analysis. If all of
the changes exclusively follow the Lambert–Beer law, the spectra should not exhibit any asynchronous properties. however, well-observed asynchronous peaks have
shown specific spectral changes caused by an increase in ovalbumin concentration
from 2 to 8 % by weight (wt) [90]. these changes were a good spectral signature
of the structural changes caused by the water-mediated protein–protein intermolecular interaction. Analysis of the synchronous and asynchronous maps generated
from the concentration-dependent spectra measured in a broad temperature range
has allowed the concentration-controlled interaction to be monitored as the protein
structure evolved from the native to the denatured unfolded state. the use of nearIR spectra in a range of overtone and combination bands attributed to water and
protein, respectively, has made it possible to analyze the role of hydration water in
the protein–protein intermolecular interactions at different stages of thermal denaturation. It has been shown that the hydration of ovalbumin is almost unchanged
from 45 to 67 °C; in this range ovalbumin molecules were in a natively folded state.
A sudden change in the hydration was observed in the narrow temperature range of
67–69 °C, in which the unfolding of the ordered secondary structures started. the
hydration again remained nearly unchanged upon further heating to 80 °C.
the nonspecific spectral changes induced by concentration are rather weak compared with those generated by other types of perturbations. thus, especially in the
case of IR-AtR measurements, it should be checked the influence of the interaction
of the protein with the surface of the internal reflection element (IRE); this interac-
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
structure, which preceded the aggregation process. Additionally, an increased number of β-turns at the expense of β-sheet content was reported. That study confirmed
the potential of 2d-IR-CoS spectroscopy to investigate the intrinsic temperaturemediated or direct lipid-mediated change in protein conformation in a lipid bilayer
environment.
It is outside of the scope of this article to mention all of the results in which
temperature perturbation has been applied in 2d infrared correlation spectroscopy.
the selected examples are intended to illustrate that there is only one step between
the very common near-IR/mid-IR temperature-dependent experiment and 2d correlation spectroscopy. Why should this step be taken? A clear answer arises from the
above examples: In contrast to ordinary one-dimensional analysis, two-dimensional
infrared correlation spectroscopy can provide insight at the molecular level into the
underlying mechanisms responsible for protein unfolding, including protein aggregation and fibrillation. Can we expect anything more from a routine temperaturedependent IR experiment?
8.3.3 Concentration of protein
Concentration has been a commonly used perturbation in investigations by ozaki
and co-workers. variations in the concentration of protein at a given temperature
have been the main source of spectral changes subjected to 2dCoS analysis. If all of
the changes exclusively follow the Lambert–Beer law, the spectra should not exhibit any asynchronous properties. however, well-observed asynchronous peaks have
shown specific spectral changes caused by an increase in ovalbumin concentration
from 2 to 8 % by weight (wt) [90]. these changes were a good spectral signature
of the structural changes caused by the water-mediated protein–protein intermolecular interaction. Analysis of the synchronous and asynchronous maps generated
from the concentration-dependent spectra measured in a broad temperature range
has allowed the concentration-controlled interaction to be monitored as the protein
structure evolved from the native to the denatured unfolded state. the use of nearIR spectra in a range of overtone and combination bands attributed to water and
protein, respectively, has made it possible to analyze the role of hydration water in
the protein–protein intermolecular interactions at different stages of thermal denaturation. It has been shown that the hydration of ovalbumin is almost unchanged
from 45 to 67 °C; in this range ovalbumin molecules were in a natively folded state.
A sudden change in the hydration was observed in the narrow temperature range of
67–69 °C, in which the unfolding of the ordered secondary structures started. the
hydration again remained nearly unchanged upon further heating to 80 °C.
the nonspecific spectral changes induced by concentration are rather weak compared with those generated by other types of perturbations. thus, especially in the
case of IR-AtR measurements, it should be checked the influence of the interaction
of the protein with the surface of the internal reflection element (IRE); this interac-