219
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
absorption band and in the series of spectra, and effects related to the wavelength
dependence of the penetration depth [125] should be reduced.
usually, in the final stage of the pretreatment process, the spectra must be normalized. this procedure is necessary if there is a strong contribution from the nonspecific variance and a weak contribution from the specific variance. Skipping
this step could lead to a loss of information from the synchronous spectra and the
false interpretation of the asynchronous spectra. Normalization allows one to isolate frequency-specific spectral variations arising from secondary structure changes
from those nonspecific spectral variations shaped by ordinary effects such as density or concentration changes. Finally, when the well-separated specific changes
are processed by 2dCoS, the changes can be converted into the different structural
events that shape the folding funnel. one of the most serious problems in 2d correlation spectroscopy is the appearance of artifacts in the 2d maps; these artifacts
could result from pretreatment steps that are incorrectly performed or omitted and
from improperly selected normalization [84].
8.3.2 External Perturbations
there are numerous physical and chemical perturbations that have been used to
induce a specific spectral response, which is the unique answer of the different
structural elements of the biomolecules to the applied stimulation. the most popular
perturbations in protein studies are temperature, h/d exchange, pressure, protein
concentration, ph, and chemical denaturants.
8.3.2.1 Temperature
In the field of protein research through 2dCoS, temperature is the most commonly
used static perturbation. temperature changes are commonly used because ordinary
changes in density, which give rise to nonspecific spectral changes, usually are accompanied by structural changes in the protein backbone upon heating or cooling. For a range of amide bands, these changes are manifested by specific spectral
variations that are well suited to the 2dCoS analysis. one of the first such studies
concerned the investigation of the order of intensity changes and the prediction of
the sequence of structural evolution during the temperature-induced unfolding processes of the ribonuclease A (RNase A) and λ Cro-V55C repressor proteins [38, 50].
the aim of the research was to determine whether the folding/unfolding process is
fully cooperative or whether the process proceeds through sequential elements. to
address this question, it was necessary to use tools that could resolve the amide I
envelope into components that could be assigned to different events. Being aware
of the facts presented in [4], mantsch’s group has turned to the 2dCoS approach,
which provides a perfect method to improve the resolution of the individual band
components contributing to the amide I envelope and which facilitates the detection
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
absorption band and in the series of spectra, and effects related to the wavelength
dependence of the penetration depth [125] should be reduced.
usually, in the final stage of the pretreatment process, the spectra must be normalized. this procedure is necessary if there is a strong contribution from the nonspecific variance and a weak contribution from the specific variance. Skipping
this step could lead to a loss of information from the synchronous spectra and the
false interpretation of the asynchronous spectra. Normalization allows one to isolate frequency-specific spectral variations arising from secondary structure changes
from those nonspecific spectral variations shaped by ordinary effects such as density or concentration changes. Finally, when the well-separated specific changes
are processed by 2dCoS, the changes can be converted into the different structural
events that shape the folding funnel. one of the most serious problems in 2d correlation spectroscopy is the appearance of artifacts in the 2d maps; these artifacts
could result from pretreatment steps that are incorrectly performed or omitted and
from improperly selected normalization [84].
8.3.2 External Perturbations
there are numerous physical and chemical perturbations that have been used to
induce a specific spectral response, which is the unique answer of the different
structural elements of the biomolecules to the applied stimulation. the most popular
perturbations in protein studies are temperature, h/d exchange, pressure, protein
concentration, ph, and chemical denaturants.
8.3.2.1 Temperature
In the field of protein research through 2dCoS, temperature is the most commonly
used static perturbation. temperature changes are commonly used because ordinary
changes in density, which give rise to nonspecific spectral changes, usually are accompanied by structural changes in the protein backbone upon heating or cooling. For a range of amide bands, these changes are manifested by specific spectral
variations that are well suited to the 2dCoS analysis. one of the first such studies
concerned the investigation of the order of intensity changes and the prediction of
the sequence of structural evolution during the temperature-induced unfolding processes of the ribonuclease A (RNase A) and λ Cro-V55C repressor proteins [38, 50].
the aim of the research was to determine whether the folding/unfolding process is
fully cooperative or whether the process proceeds through sequential elements. to
address this question, it was necessary to use tools that could resolve the amide I
envelope into components that could be assigned to different events. Being aware
of the facts presented in [4], mantsch’s group has turned to the 2dCoS approach,
which provides a perfect method to improve the resolution of the individual band
components contributing to the amide I envelope and which facilitates the detection