Gaining insight into protein structure via ATR-FTIR spectroscopy

Abstract

During the last decades, Fourier-transform infrared spectroscopy (FTIR) with attenuated total reflectance (ATR) has gained a substantial role in monitoring structural changes of proteins. The conformation of the polypeptide backbone, reflecting the pattern of intramolecular hydrogen bonding, affects the vibrational energy of carbonyl groups and, consequently, the absorption of infrared light. Specifically sensitive to the conformational state of a polypeptide is the Amide I region (1700–1600 cm⁻¹), whose individual bands correlate with distinct secondary structures. ATR-FTIR thus provides the possibility to determine secondary structure content by deconvolution of the Amide I region, which makes it a valuable tool for investigating protein structure. Furthermore, the sensitivity of the Amide I band to subtle differences in hydrogen bonding enables discrimination between different β-sheet conformations, including intramolecular and intermolecular β-sheet. Compared to other methods for secondary structure determination, such as circular dichroism, this advantage makes infrared spectroscopy an excellent tool for monitoring aggregation processes. This review is intended to explain the principle of FTIR, the specificities of protein FTIR spectroscopy, to correlate the spectra with the protein secondary structure, and to provide different approaches for spectral analysis. To highlight FTIR contribution as a reliable parameter in protein structural analysis, here we review the data regarding the determination of the secondary structure of native globular proteins, the monitoring of discrete conformational changes upon destabilizing treatments, and the monitoring of structural changes in the aggregation process.