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 pro teins 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 recorde d. 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 techni ques were compared for the determination of secondary structure conten t [classical least-squares analysis (CLS) and partial least-squares an alysis (PLS)] and optimum conditions for their utilization were sugges ted. The best correlation between the ATR-FT-IR approach and X-ray dif fraction data was obtained with the PLS analysis and the distinction o f four types of secondary structures (ordered and disordered alpha-hel ix, beta-sheet and undefined conformation). The averages of the differ ences in the percentage content between X-ray and IR secondary structu res predicted in the ATR mode are 7.1% and 2.8% for the ordered and di sordered alpha-helix, respectively, 6.5% for the beta-sheet and 4.7% f or the undefined structure.