STUDY OF THE SECONDARY STRUCTURE OF PROTEINS IN AQUEOUS-SOLUTIONS BY ATTENUATED TOTAL-REFLECTION FOURIER-TRANSFORM INFRARED SPECTROMETRY

An approach to the determination of secondary structure content in proteins in aqueous solutions based on attenuated total reflection (ATR) Fourier transform infrared (FT-IR) spectrometry is proposed. ATR-FT-IR spectra of eleven proteins with known crystal structures were recorded. An algorithm for careful subtraction of the solvent background was developed and the reproducibility of the spectra was established for a wide range of protein concentrations in aqueous solutions. Two techniques were compared for the determination of secondary structure content [classical least-squares analysis (CLS) and partial least-squares analysis (PLS)] and optimum conditions for their utilization were suggested. The best correlation between the ATR-FT-IR approach and X-ray diffraction data was obtained with the PLS analysis and the distinction of four types of secondary structures (ordered and disordered alpha-helix, beta-sheet and undefined conformation). The averages of the differences in the percentage content between X-ray and IR secondary structures predicted in the ATR mode are 7.1% and 2.8% for the ordered and disordered alpha-helix, respectively, 6.5% for the beta-sheet and 4.7% for the undefined structure.