1. Protein structure
Secondary structure prediction:
FTIR spectroscopy is a powerful tool to study protein and peptide secondary structure. Using our database of proteins for which the structure has been determined accurately by methods such as X-ray crystallography, we are able to predict the alpha-helix and beta-sheet content using the FTIR spectra of any protein.
Ultraviolet Circular dichroism (UV-CD) is one of the standard method to examine the secondary structure of proteins. However, compared to FTIR spectroscopy, this technique is less sensitive to beta-sheet structures. In addition, UV-CD cannot detect intermolecular beta-sheet structures which form upon protein aggregation. Finally, the measurement by UV-CD cannot be realized at higher concentration range, typically encountered in antibody formulations.
FTIR spectroscopy is also an exquisite tool to study and discriminate amyloid aggregates. Based on the analysis of specific spectral features of beta-sheet structures, oligomers and fibrils can be differentiated. It is a rapid and low protein material-demanding method.
Hydrogen isotope exchange has been extensively used for the analysis of protein structure and dynamics. It appears to be one of the main techniques able to identify sub molecular motional domains including fast exchanging protons of the protein surface, somewhat slower exchanging protons of the flexible (loop) regions buried in the protein or involved in some secondary structures, and the slowly exchanging protons from the protein core formed by the most rigid clusters (knots) of amino acids. The kinetics of exchange can be monitored by mass spectroscopy or by FTIR spectroscopy. FTIR data usually yield estimates of the exchange rate for the protein as a whole. When compared to mass spectrometry, the great advantage of monitoring the exchange by FTIR is that the measure is focused on the amide protons only, yielding data proportional to the number of residues in the protein. Another advantage of FTIR is that the measurements are obtained in real time (no quenching of the reaction is required). The 1H/2H exchange kinetic monitored by FTIR spectroscopy allows the characterization of the protein in different experimental conditions. Indeed, the exchange rate contains important information on the structure and the structural stability of the protein. For example, it has been shown that 1H/2H exchange kinetics allowed monitoring of the tertiary structure changes of the gastric H+/K+‐ATPase in the presence of Na+, K+, Ca2+, Ca2+ and ADP or ATP.
Goormaghtigh, E.; Ruysschaert, J.-M.; Raussens, V. Evaluation of the information content in infrared spectra for protein secondary structure determination. Biophys J 2006, 90, 2946–57.
Sarroukh, R.; Goormaghtigh, E.; Ruysschaert, J.-M.; Raussens, V. ATR-FTIR: a “rejuvenated” tool to investigate amyloid proteins. Biochim. Biophys. Acta 2013, 1828, 2328–38.
Ruysschaert, J.-M.; Raussens, V. ATR-FTIR Analysis of Amyloid Proteins. In Methods in molecular biology; 2018; Vol. 1777, pp. 69–81.
Vigano, C.; Manciu, L.; Buyse, F.; Goormaghtigh, E.; Ruysschaert, J.-M. Attenuated total reflection IR spectroscopy as a tool to investigate the structure, orientation and tertiary structure changes in peptides and membrane proteins. Biopolymers 2000, 55, 373–380