Application of Near-Infrared and Fourier Transform Infrared Spectroscopy in the Characterization of Ligand-Induced Conformation Changes in Folate Binding Protein Purified from Bovine Milk: Influence of Buffer Type and pH
Fourier transform infrared (FT-IR) and near-infrared (NIR) spectroscopy have been applied to detect structural alterations in folate binding protein (FBP) induced by ligation in different buffer types. The amide I region pointed to a β-sheet to α-helix transition upon ligation
in acetate and phosphate buffers, and the formation of intermolecular β-sheet was indicated at pH 5.0, in agreement with a dimerization of FBP taking place at this pH. The ligand-induced changes in the 2100–2300 nm NIR region were significant for FBP in acetate and phosphate buffers
of pH 5.0, and the variations were interpreted as secondary structure changes, based on previous assignments of secondary structures to the combination bands in the NIR region. In the case of acetate buffer, variations in the amide combination bands agreed with the amide I analysis, but for
the other buffer types some discrepancies were found and explained by side-chain contributions to the NIR, which could reflect the tertiary and quaternary structure differences. NIR spectra of FBP at pH 7.4 and 5.0 revealed contradictory effects on the side chains, reflecting different polymerization
events at the two pH values, whereas the amide I region indicated similar changes at the two pH values. Therefore, we suggest that FT-IR and NIR spectroscopy may complement each other, such that the two techniques in combination may give information on all three types of protein conformational
changes. While the secondary structure changes are revealed by FT-IR, the tertiary and quaternary structure changes are reflected in the NIR spectra, although the general influence of the latter changes on the NIR spectra remains to be confirmed.
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Kgs. Lyngby, Denmark 2:
Department of Clinical Chemistry, Hillerød Hospital, Hillerød, Denmark
Publication date: July 1, 2006
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