227
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
tween bands arising from vibrations of exchangeable protons from N–h amide
bonds and protons belonging to the Arg and tyr side chains. In the experiment,
subsequent spectra were acquired every 10 min for approximately 14 h, and the
simulated spectra that mimicked the changes of the experimental spectra as much
as possible were subjected to 2dCoS. the comparison of only the synchronous
spectra for the experimental and simulated data from 1,800–1,400 cm
−1
resulted in
the separation of the highly overlapped absorbances from the backbone and from
the Arg and tyr side chains. In [78], the studies were extended to next three EgFR
ligands (ER, hB-EgF, AR), and in the analysis, the asynchronous spectra were also
employed; these spectra allowed the detection of the differences in the exchange
rates for these five ligands, within the side chains and the main back-bone; these
differences may be critical for receptor regulation. other types of systems for which
very detailed assignments of different structural elements to sequential changes in
the h/d exchange have been presented are centrin [66] and the complex of centrin
with melittin [85].
8.3.5 Pressure
Smeller and heremans [45] first applied 2dCoS to analyze the pressure-induced
changes in the FtIR spectra of proteins. Initially, a small protein such as bovine
pancreatic trypsin inhibitor (BPtI, 8 kda) was subjected to h/d exchange overnight at room temperature. Next, a high pressure (0–1 gPa) was applied as a perturbation, and the FtIR spectra were measured. the application of 2dCoS has been
very efficient in the separation of the spectral features arising from h/d exchange
from those spectral features that are pressure-induced. In the low pressure region
(0–0.3 gPa), both conformational and exchange effects took place whereas above
0.5 gPa (up to 0.9 gPa), the exchange was complete and only conformational
changes were observed [46]. the next problem of interest was the comparison of
this small protein with lipoxygenase (LoX), which is a large protein (102 kda).
the application of 2dCoS helped unravel the relation between the conformational
changes and the kinetics of the h/d exchange processes for the two types of proteins [51]. the pressure range was divided into three parts for the 2d correlation
analysis: the low pressure range (2–4 kbar), the transition region (4.7–9.0 kbar), and
the upper pressure region (8.5–10.6 kbar). It has been observed that for BPtI, the
low pressure regime acts preferentially on the conformational dynamics and promotes h/d exchange. For LoX, the pressure induced irreversible conformational
changes, which were responsible for increased exposure of the interior parts of the
protein to the solvent.
Poly(L-lysine) (PLL) is a very attractive polypeptide because under different
conditions, it can form one of three stable conformations: an α-helix, an anti-parallel β-sheet, or a random coil. The pressure resistance of 70 kDa PLL has been
checked over a pressure increase from 0.1 to 1,150 mPa [87]. guided by PCA results, the pressure dependent spectra have been separated into low (0.1–400 mPa)
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
tween bands arising from vibrations of exchangeable protons from N–h amide
bonds and protons belonging to the Arg and tyr side chains. In the experiment,
subsequent spectra were acquired every 10 min for approximately 14 h, and the
simulated spectra that mimicked the changes of the experimental spectra as much
as possible were subjected to 2dCoS. the comparison of only the synchronous
spectra for the experimental and simulated data from 1,800–1,400 cm
−1
resulted in
the separation of the highly overlapped absorbances from the backbone and from
the Arg and tyr side chains. In [78], the studies were extended to next three EgFR
ligands (ER, hB-EgF, AR), and in the analysis, the asynchronous spectra were also
employed; these spectra allowed the detection of the differences in the exchange
rates for these five ligands, within the side chains and the main back-bone; these
differences may be critical for receptor regulation. other types of systems for which
very detailed assignments of different structural elements to sequential changes in
the h/d exchange have been presented are centrin [66] and the complex of centrin
with melittin [85].
8.3.5 Pressure
Smeller and heremans [45] first applied 2dCoS to analyze the pressure-induced
changes in the FtIR spectra of proteins. Initially, a small protein such as bovine
pancreatic trypsin inhibitor (BPtI, 8 kda) was subjected to h/d exchange overnight at room temperature. Next, a high pressure (0–1 gPa) was applied as a perturbation, and the FtIR spectra were measured. the application of 2dCoS has been
very efficient in the separation of the spectral features arising from h/d exchange
from those spectral features that are pressure-induced. In the low pressure region
(0–0.3 gPa), both conformational and exchange effects took place whereas above
0.5 gPa (up to 0.9 gPa), the exchange was complete and only conformational
changes were observed [46]. the next problem of interest was the comparison of
this small protein with lipoxygenase (LoX), which is a large protein (102 kda).
the application of 2dCoS helped unravel the relation between the conformational
changes and the kinetics of the h/d exchange processes for the two types of proteins [51]. the pressure range was divided into three parts for the 2d correlation
analysis: the low pressure range (2–4 kbar), the transition region (4.7–9.0 kbar), and
the upper pressure region (8.5–10.6 kbar). It has been observed that for BPtI, the
low pressure regime acts preferentially on the conformational dynamics and promotes h/d exchange. For LoX, the pressure induced irreversible conformational
changes, which were responsible for increased exposure of the interior parts of the
protein to the solvent.
Poly(L-lysine) (PLL) is a very attractive polypeptide because under different
conditions, it can form one of three stable conformations: an α-helix, an anti-parallel β-sheet, or a random coil. The pressure resistance of 70 kDa PLL has been
checked over a pressure increase from 0.1 to 1,150 mPa [87]. guided by PCA results, the pressure dependent spectra have been separated into low (0.1–400 mPa)