225
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
8.3.4 H/D exchange
hydrogen–deuterium (h/d) exchange of the amide protons has found important
applications in many experimental studies of protein structure. the contributions
of different secondary structures to the amide bands can be efficiently separated
because of the different exchange rates, which are controlled by the solvent accessibility and hydrogen bond stability of the amide protons [129]. Infrared measurements as a function of time collected a large data set dominated by specific spectral
changes in the range of the amide I, amide I’, and amide II’ bands; these changes
make h/d exchange an excellent external perturbant.
the advantage of this perturbation in the 2dCoS analysis was first shown in
Pézolet’s group for an aqueous solution of myoglobin measured by IR-AtR spectroscopy [36]. Because of the different rates of the h/d exchange for different elements of the secondary structure of myoglobin, this method has allowed the identification of at least five components of the amide I band; these components were
assigned to the α-helix, intermolecular β-sheet, β-turn, and random coil conformations. these results have initiated many investigations in which h/d exchange has
been chosen as a perturbation. this methodology has been applied to monitor the
changes in secondary structure of a small basic protein found in the endosperm
of wheat seeds (β-purothionin) because of the incorporation of this protein into
lipid bilayers composed of dimyristoylphosphatidylglycerol (dmPg). Comparative analysis of the 2dCoS synchronous and asynchronous maps allowed the observation of an increase in the α-helix content of β-purothionin; this increase has
confirmed the formation of a functional protein channel in the membrane upon the
binding of the protein to the lipid membrane [67].
this effective method of unraveling the different components in the poorly resolved amide I, II, and II′ bands of proteins was also used by Meskers et al. [4443]
to determine whether the h/d exchange process in streptavidin was modified in
the presence of biotin. generally, the binding of a ligand can be accompanied by
conformational changes at the protein site that can propagate throughout the entire
protein. therefore, the analysis was focused on distinguishing specific absorbance
variations in the 1,400–1,700 cm
−1
range and on the assignment of these variations to subtle differences within the secondary structure. 2dCoS results have revealed that the strong non-covalent interactions between streptavidin and the biotin
ligand inhibited the exchange for some of the amide protons, which were part of
the β-sheet structure of this protein. This inhibition was identified from the slowly
proceeding changes at 1,530 and 1,445 cm
−1
, which were assigned to the amide II
and II′ frequencies of the β-sheet.
Wu et al. [57] used 2d-IR-CoS supported by PCA to investigate the kinetics
of h/d exchange in human serum albumin (hSA). From the PCA results, the exchange process has been separated into three stages that have different structural
change profiles, as shown by the loadings. the slice and power spectra extracted
from the synchronous and asynchronous maps were independently calculated for
the data grouped into the three time domains. the results have shown that during
8 Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein Research
8.3.4 H/D exchange
hydrogen–deuterium (h/d) exchange of the amide protons has found important
applications in many experimental studies of protein structure. the contributions
of different secondary structures to the amide bands can be efficiently separated
because of the different exchange rates, which are controlled by the solvent accessibility and hydrogen bond stability of the amide protons [129]. Infrared measurements as a function of time collected a large data set dominated by specific spectral
changes in the range of the amide I, amide I’, and amide II’ bands; these changes
make h/d exchange an excellent external perturbant.
the advantage of this perturbation in the 2dCoS analysis was first shown in
Pézolet’s group for an aqueous solution of myoglobin measured by IR-AtR spectroscopy [36]. Because of the different rates of the h/d exchange for different elements of the secondary structure of myoglobin, this method has allowed the identification of at least five components of the amide I band; these components were
assigned to the α-helix, intermolecular β-sheet, β-turn, and random coil conformations. these results have initiated many investigations in which h/d exchange has
been chosen as a perturbation. this methodology has been applied to monitor the
changes in secondary structure of a small basic protein found in the endosperm
of wheat seeds (β-purothionin) because of the incorporation of this protein into
lipid bilayers composed of dimyristoylphosphatidylglycerol (dmPg). Comparative analysis of the 2dCoS synchronous and asynchronous maps allowed the observation of an increase in the α-helix content of β-purothionin; this increase has
confirmed the formation of a functional protein channel in the membrane upon the
binding of the protein to the lipid membrane [67].
this effective method of unraveling the different components in the poorly resolved amide I, II, and II′ bands of proteins was also used by Meskers et al. [4443]
to determine whether the h/d exchange process in streptavidin was modified in
the presence of biotin. generally, the binding of a ligand can be accompanied by
conformational changes at the protein site that can propagate throughout the entire
protein. therefore, the analysis was focused on distinguishing specific absorbance
variations in the 1,400–1,700 cm
−1
range and on the assignment of these variations to subtle differences within the secondary structure. 2dCoS results have revealed that the strong non-covalent interactions between streptavidin and the biotin
ligand inhibited the exchange for some of the amide protons, which were part of
the β-sheet structure of this protein. This inhibition was identified from the slowly
proceeding changes at 1,530 and 1,445 cm
−1
, which were assigned to the amide II
and II′ frequencies of the β-sheet.
Wu et al. [57] used 2d-IR-CoS supported by PCA to investigate the kinetics
of h/d exchange in human serum albumin (hSA). From the PCA results, the exchange process has been separated into three stages that have different structural
change profiles, as shown by the loadings. the slice and power spectra extracted
from the synchronous and asynchronous maps were independently calculated for
the data grouped into the three time domains. the results have shown that during