Structural characterization of polypeptides by Fourier transform infrared (FT-IR) spectroscopy is based on the absorption of infrared light by the amide backbone function (reviewed by Susi and Byler, 1986; Jackson and Mantsch, 1995). The amide I band, within the 1600-1700 cm-1 frequency region of IR spectra, is most commonly used for secondary structure determination, and contains contributions from C=O stretching vibration of amide groups (coupled to the in-phase bending of the N-H bond and the C-N stretching vibration). Due to a strong O-H bending absorption of water at 1644 cm-1, deuterated water (D2O) is frequently used for FT-IR analysis. The annotation amide I’ is used when analysing deuterated proteins. Because the amide groups of the polypeptide backbone are involved in secondary structure interactions of different types, separate amide I’ absorptions are observed that are characteristic for elements of secondary structure (Byler and Susi, 1986; Table 1). Because band widths of each of these components are greater than the instrument resolution, the amide I’ band of proteins typically is a composite of overlapping sub-components. A number of mathematical techniques have been developed that allow visualization of overlapping bands following manipulation of the spectrum (reviewed by Surewicz et al., 1993).
Table: Assignment of secondary structure components to characteristic frequencies in the amide I’ band of FT-IR spectra of globular proteins (Byler and Susi, 1986).
Element of Deconvolved amide I’ Root mean square
secondary structure frequency (cm-1) deviation
Extended chains 1624 2.4
(
Unordered 1645 1.6
Helix 1654 1.5
Turns and Bends 1663 2.2
1670 1.4
1683 1.5
1688 1.1
1694 1.7
The assignments are based on the deconvolved FT-IR spectra of 21 globular proteins with known structure.
Samples of CAT were resuspended in D2O buffered with 0.01 M Bis-Tris (pD 7.0). H-to-D exchange occurred at room temperature during at least 16 h before the infrared spectroscopic measurements. For the temperature denaturation studies, a CaF2 cell with 50 µm spacer was filled with CAT solutions of approximately 100 mg/ml. The temperature of the sample was regulated by an automatic temperature controller (Graseby Specac Ltd., Kent, UK). Infrared spectra were obtained with a Bruker IFS66 FTIR spectrometer (Karlsruhe, Germany) equipped with a broad band mercury-cadmium-telluride (MCT) detector. The infrared light was focused on the sample by a NaCl lens. For each spectrum 350 interferograms were co-added and Fourier-transformed to give a resolution of 2 cm-1.