Conformational Changes

Protein conformation is of paramount importance in understanding biological materials. Changes in protein conformation may occur with only relatively small amounts of energy, e.g. when applying shear. Such conformational dynamics of protein molecules is best investigated by a rheometer since it can apply defined amounts of shear to a sample to simulate almost any shear process.

Traditionally, the sample is then be removed after shearing to determine and quantify the structure by FTIR spectroscopy. In particular, the shape and frequency of the amide I band, which is assigned to the C=O stretching vibration within the peptide bonds of the protein molecule, is very characteristic for the structure of the analyzed protein. From this absorption band the presence and quantity of secondary structure elements (like alpha-helix, beta-sheet) in a protein can be determined and conformational changes are detected with high sensitivity. Any alterations in the structure regardless if they were induced by temperature or shear, ligand binding, mutations etc. are reflected by specific absorptive changes in the amide I region. Furthermore, side chains of various amino acids can be monitored and directly compared under varying environmental conditions, or in mutant proteins with altered primary sequence.

However, in the same way as a rheometer cannot detect conformational changes directly and in situ, FTIR spectroscopy cannot detect transient changes as long as such techniques are applied separately. Therefore, to comprehensively understand the mechanical properties of such materials it requires combining the macroscopic rheological approach with FTIR spectroscopy to apply them simultaneously on one and the same sample under exactly the same conditions.

Based on this principle, the RHEONAUT provides not only the simultaneous collection of IR spectra, but in synchrony with rheological data to adress key questions at the molecular level.

To summarize, the RHEONAUT is well qualified to monitor shear induced unfolding, refolding and denaturation processes of proteins, as well as the formation of multimeric structures like aggregates or fibrils, the impact of ligand-binding, and more in a temperature range between 0 – 100 °C even if such changes are fully reversible.