Application of multivariate curve resolution to the temperature-induced unfolding of α-chymotrypsin

Abstract

Thermal-induced unfolding of α-chymotrypsin has been monitored with circular dichroism spectroscopy, which shows a far-UV-CD region sensitive to changes in the protein secondary structure and a near-UV-CD region, which gives information at the tertiary structure level. Changes in CD signals in both the far-UV and the near-UV are used to monitor comprehensively the loss of protein structure during unfolding. The application of the chemometric method multivariate curve resolution–alternating least-squares (MCR–ALS) to the spectroscopic measurements allowed for the recovery of the concentration profiles and spectra of three different protein conformations, one of them not obtainable experimentally. Joining the resolved information about the evolution of the tertiary structure and the results coming from methods devoted to the elucidation of the protein secondary structure, the three protein conformations can be characterised as: a native conformation, with both secondary and tertiary structure organized as in the natural active protein; a second conformation, with a modified secondary structure richer in β-sheet and a native-like tertiary structure, and a third conformation, with a secondary structure very similar to the second conformation and with the tertiary structure unfolded.