Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy

This protocol describes how in-solution protein FTIR can be used to obtain information about the relative contributions of α-helices, β-sheets, β-turn, and random coil structures to a protein's secondary structure. Fourier transform IR (FTIR) spectroscopy is a nondestructive technique for structural characterization of proteins and polypeptides. The IR spectral data of polymers are usually interpreted in terms of the vibrations of a structural repeat. The repeat units in proteins give rise to nine characteristic IR absorption bands (amides A, B and I–VII). Amide I bands (1,700–1,600 cm −1 ) are the most prominent and sensitive vibrational bands of the protein backbone, and they relate to protein secondary structural components. In this protocol, we have detailed the principles that underlie the determination of protein secondary structure by FTIR spectroscopy, as well as the basic steps involved in protein sample preparation, instrument operation, FTIR spectra collection and spectra analysis in order to estimate protein secondary-structural components in aqueous (both H 2 O and deuterium oxide (D 2 O)) solution using algorithms, such as second-derivative, deconvolution and curve fitting. Small amounts of high-purity (>95%) proteins at high concentrations (>3 mg ml −1 ) are needed in this protocol; typically, the procedure can be completed in 1–2 d.