A new attenuated total reflectance Fourier transform infrared spectroscopy method for the study of proteins in...

An attenuated total reflectance Fourier transform infrared method has been developed that allows collection of spectra from proteins in solution. This method eliminates any structural perturbations induced by the internal reflection element (IRE), and thus the spectra reflect the solution conformation of the protein. A key feature of the method is subtraction of the signal from any protein adsorbed to the IRE. The advantages of this method include the small amount of sample required and the high sampling rate. Attenuated total reflectance (ATR)-Fourier transform infrared spectroscopy (FTIR) is more versatile than transmission FTIR because it is possible to collect spectra of nontransparent samples, to use samples of very low protein concentration (< or = 0.3 mg/ml), and to study proteins in the presence of strongly absorbing solutes (such as denaturants). The experimental procedures and data processing routines developed were evaluated by collecting spectra from a set of 13 proteins and evaluating their accuracy with a partial least-squares analysis. The relative mean and standard deviation errors for the basis set analysis were 6.3% for alpha-helix, 5.9% for beta-sheet/extended structure, and 4.4% for turn, which are similar to values from comparable analyses of transmission FTIR spectra. In addition, a detailed comparison between this solution ATR method and the hydrated thin-film ATR technique is presented.