229
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
1,696 cm
−1
, which arise from the C = o stretching modes of the free Cooh groups
and the Cooh groups involved in hydrogen bonds of increasing strength. this
analysis strongly supported the hypothesis that protonation of the Coo
−
groups of
the glutamic and aspartic acid residues has occurred prior to the structural changes
that develop in the α-helices and that dominate in HSA folding. The N–F transition
has been correlated with the presence of free Cooh groups and the formation of
medium-strength hydrogen bonds between the Cooh groups and groups from the
main chain, side chains or water molecules. the asynchronous spectrum confirmed
that the hydrogen bonds play a key role in the initiation of the N–F transition and
trigger the α-helical changes specific to the transition. The synchronous and asynchronous spectra of the F form have shown that the β-strands and β-turns of HSA
change significantly in the ph region between 3.8 and 3.0.
2dCoS has been applied to ph-perturbed mid-IR spectra of porcine plasma proteins [82]. the system has a variety of proteins with diverse secondary structure,
and when subjected to a ph decrease from 7.5 to 4.5, the proteins undergo both
unfolding and aggregation processes at 30 °C. A detailed analysis of the sequence
of spectral intensity changes that take place during the ph decrease has been performed for six peaks assigned to the most common secondary structure elements
of the proteins present in the plasma. the studies have shown that in the denaturation process that begins at a higher ph, the globulin fraction is primarily involved
whereas the serum albumin fraction is denatured under more acidic conditions. ph
perturbation was also used in the first NmR-Raman heterospectral correlation studies of the structural evolution of silk fibroin [104].
8.3.7 Other Types of Perturbations
the properties of silk fiber from Bombyx mori have been analyzed by dynamic
step-scan FtIR measurements as a function of a sinusoidal mechanical strain [52].
the dynamic spectra revealed that the stress-induced dynamic reorientation in the
fibroin film was mainly localized in the segment with a β-sheet conformation and
was almost synchronous with the applied mechanical strain. Increased surface pressure has been a common perturbation in studies conducted by dluhy’s group, and
infrared reflection–absorbance spectroscopy was used for the measurements [35,
58, 68].
2dCoS was suitable for the real-time detection of the molecular processes occurring in an intact retina as a response to light [79]. 2dCoS has facilitated the
identification of bands arising from the same molecular chromophores even in the
presence of very complex spectral changes. A process in which a β-sheet-rich protein is released from the disk membrane and is redistributed from the outer segment
towards the inner segment of rod cells has been postulated.
A varying concentration of denaturing agent has been used as a perturbation in
the analysis of the influence of bromoethanol on the structure of β-lactoglobulin
studied by IR in both ranges [37]. two different pictures of sequential changes
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
1,696 cm
−1
, which arise from the C = o stretching modes of the free Cooh groups
and the Cooh groups involved in hydrogen bonds of increasing strength. this
analysis strongly supported the hypothesis that protonation of the Coo
−
groups of
the glutamic and aspartic acid residues has occurred prior to the structural changes
that develop in the α-helices and that dominate in HSA folding. The N–F transition
has been correlated with the presence of free Cooh groups and the formation of
medium-strength hydrogen bonds between the Cooh groups and groups from the
main chain, side chains or water molecules. the asynchronous spectrum confirmed
that the hydrogen bonds play a key role in the initiation of the N–F transition and
trigger the α-helical changes specific to the transition. The synchronous and asynchronous spectra of the F form have shown that the β-strands and β-turns of HSA
change significantly in the ph region between 3.8 and 3.0.
2dCoS has been applied to ph-perturbed mid-IR spectra of porcine plasma proteins [82]. the system has a variety of proteins with diverse secondary structure,
and when subjected to a ph decrease from 7.5 to 4.5, the proteins undergo both
unfolding and aggregation processes at 30 °C. A detailed analysis of the sequence
of spectral intensity changes that take place during the ph decrease has been performed for six peaks assigned to the most common secondary structure elements
of the proteins present in the plasma. the studies have shown that in the denaturation process that begins at a higher ph, the globulin fraction is primarily involved
whereas the serum albumin fraction is denatured under more acidic conditions. ph
perturbation was also used in the first NmR-Raman heterospectral correlation studies of the structural evolution of silk fibroin [104].
8.3.7 Other Types of Perturbations
the properties of silk fiber from Bombyx mori have been analyzed by dynamic
step-scan FtIR measurements as a function of a sinusoidal mechanical strain [52].
the dynamic spectra revealed that the stress-induced dynamic reorientation in the
fibroin film was mainly localized in the segment with a β-sheet conformation and
was almost synchronous with the applied mechanical strain. Increased surface pressure has been a common perturbation in studies conducted by dluhy’s group, and
infrared reflection–absorbance spectroscopy was used for the measurements [35,
58, 68].
2dCoS was suitable for the real-time detection of the molecular processes occurring in an intact retina as a response to light [79]. 2dCoS has facilitated the
identification of bands arising from the same molecular chromophores even in the
presence of very complex spectral changes. A process in which a β-sheet-rich protein is released from the disk membrane and is redistributed from the outer segment
towards the inner segment of rod cells has been postulated.
A varying concentration of denaturing agent has been used as a perturbation in
the analysis of the influence of bromoethanol on the structure of β-lactoglobulin
studied by IR in both ranges [37]. two different pictures of sequential changes