235
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
and combination bands from lysozyme and sucrose has been examined by connecting the NIR spectra, which are composed of rather broad and overlapping bands,
to the Raman spectra, which are composed of sharp, clearly separated and wellassigned bands. the analysis has been restricted to statistical 2d correlation and to
slice spectra extracted at specific frequencies of sucrose or lysozyme. generally,
the slice spectrum is obtained by selecting the frequency of a well-defined vibrational mode in one region whereas the frequencies in the other region change. the
spectrum contains a large amount of information about the relationship between one
selected absorber in a region and all of the absorbers in another region. Slice spectra
extracted both from the synchronous and asynchronous spectra could be very useful
in the interpretation of hetero 2dCoS results.
For the first time, the asynchronous foundations have been applied in an associated analysis of the near-IR ↔ mid-IR spectra of RNase A [50]. the lack or
presence of asynchronous heterocorrelation cross peaks between the mid-IR amide
I components assigned to specific secondary structures and the N–h combination
bands in the near-IR range determined whether the near-IR bands could have resulted from the same secondary structure.
The heterospectral correlations between the mid-IR ↔ Raman [40, 41], ECd
↔ Raman [42], and mid-IR ↔ VCD [43] spectra of the protein system have been
very attractive from the perspective of better understanding the correlation between
the bands attributed to the same secondary structure elements in the two types of
spectroscopy. heterospectral correlation has greatly facilitated band assignments,
particularly in situations in which a band was well identified in one range/spectroscopy because of a high structural sensitivity but its counterpart could not be
easily disclosed in the second range or by the second spectroscopy because of lower
optical activity, caused by differences in the selection rules [42, 43]. The mid-IR ↔
Raman 2dCoS has provided new insight into the correlation between the IR and
Raman bands in the amide III region and has confirmed the band assignments for
the unordered form [40]
Raman and RoA, two spectroscopies that are extremely sensitive to changes in
secondary structure, were pooled together to analyze the pH-induced α-helix-todisordered transition in poly(L-glutamic acid) (PLg) [105] and the temperatureinduced α-helix-to-β-sheet transition in poly(L-lysine) [108]. this combination
enabled the complete monitoring of structural changes and of spectral changes in a
much wider range of bands (from 1,800 to 800 cm
−1
) than has been practiced in the
standard analysis based only on one spectroscopy without the support of a second
one. the associated analysis presented the great potential for the discovery of new
transition mechanisms in relatively complex biological systems. It has been shown
that the ph-induced conformational change in PLg might not be a simple twostate transition from the α-helix to the disordered structure. The apparent inverse
order effect was observed during the temperature-induced transition of PLL from
an α-helix to a β-sheet. The changes in the β-sheet structure apparently occurred
before those in the α-helix. This phenomenon has been the subject of detailed studies performed with the support of a new development in 2DCoS, i.e., kν-correlation
analysis [31, 32].
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
and combination bands from lysozyme and sucrose has been examined by connecting the NIR spectra, which are composed of rather broad and overlapping bands,
to the Raman spectra, which are composed of sharp, clearly separated and wellassigned bands. the analysis has been restricted to statistical 2d correlation and to
slice spectra extracted at specific frequencies of sucrose or lysozyme. generally,
the slice spectrum is obtained by selecting the frequency of a well-defined vibrational mode in one region whereas the frequencies in the other region change. the
spectrum contains a large amount of information about the relationship between one
selected absorber in a region and all of the absorbers in another region. Slice spectra
extracted both from the synchronous and asynchronous spectra could be very useful
in the interpretation of hetero 2dCoS results.
For the first time, the asynchronous foundations have been applied in an associated analysis of the near-IR ↔ mid-IR spectra of RNase A [50]. the lack or
presence of asynchronous heterocorrelation cross peaks between the mid-IR amide
I components assigned to specific secondary structures and the N–h combination
bands in the near-IR range determined whether the near-IR bands could have resulted from the same secondary structure.
The heterospectral correlations between the mid-IR ↔ Raman [40, 41], ECd
↔ Raman [42], and mid-IR ↔ VCD [43] spectra of the protein system have been
very attractive from the perspective of better understanding the correlation between
the bands attributed to the same secondary structure elements in the two types of
spectroscopy. heterospectral correlation has greatly facilitated band assignments,
particularly in situations in which a band was well identified in one range/spectroscopy because of a high structural sensitivity but its counterpart could not be
easily disclosed in the second range or by the second spectroscopy because of lower
optical activity, caused by differences in the selection rules [42, 43]. The mid-IR ↔
Raman 2dCoS has provided new insight into the correlation between the IR and
Raman bands in the amide III region and has confirmed the band assignments for
the unordered form [40]
Raman and RoA, two spectroscopies that are extremely sensitive to changes in
secondary structure, were pooled together to analyze the pH-induced α-helix-todisordered transition in poly(L-glutamic acid) (PLg) [105] and the temperatureinduced α-helix-to-β-sheet transition in poly(L-lysine) [108]. this combination
enabled the complete monitoring of structural changes and of spectral changes in a
much wider range of bands (from 1,800 to 800 cm
−1
) than has been practiced in the
standard analysis based only on one spectroscopy without the support of a second
one. the associated analysis presented the great potential for the discovery of new
transition mechanisms in relatively complex biological systems. It has been shown
that the ph-induced conformational change in PLg might not be a simple twostate transition from the α-helix to the disordered structure. The apparent inverse
order effect was observed during the temperature-induced transition of PLL from
an α-helix to a β-sheet. The changes in the β-sheet structure apparently occurred
before those in the α-helix. This phenomenon has been the subject of detailed studies performed with the support of a new development in 2DCoS, i.e., kν-correlation
analysis [31, 32].