Fusion of incomplete QCL-IR and ECD datasets using MCR-ALS to extend the viable concentration range for studying...

While several techniques exist to study the secondary structure of proteins, mid-IR and electronic circular dichroism spectroscopy are complementary techniques that stand out for liquid measurements due to their non-destructive nature, broad applicability and ease of use. CD spectroscopy however is only used to measure low protein concentrations and suffers from signal interferences from common buffers. With advances in laser-technology, the achievable pathlengths used in IR-spectroscopy have increased with the use of external cavity quantum cascade lasers (EC-QCL) as light sources. This has not only enabled protein denaturation to be studied without aggregates clogging the measurement cell, as was the case with shorter pathlengths, but also expanded the concentration range that can be measured with the technique. Yet, the concentration range where both IR and CD measurements can be made has only a small overlap. In this study, the IR and CD spectra obtained during the thermal denaturation of α-chymotrpysin (α-CT) in the overlapping concentration range of the two spectroscopy techniques, between 10mgmL^-1 to 40mgmL^-1, are used to extract more information about the denaturation process of the protein and its concentration dependence. To this end, the protein denaturation spectra from both methods between 20°C to 80°C are fused and analyzed using multivariate curve resolution-alternating least squares (MCR-ALS), a bilinear unmixing technique that provides thermal profiles and pure fingerprints of the protein conformations involved in the denaturation process. However, in these measurements, a large and information-dense part of the CD spectra found at the lowest UV wavelengths cannot be used due to the saturated signal linked to high protein concentration levels. A modified version of MCR-ALS is hence introduced to overcome this issue, which allows all available information to be used without sacrificing the quality of the fit. The prowess of the adapted MCR-ALS technique as a tool for data fusion was demonstrated by not only providing a better fit than the individual models (CD and IR spectra analyzed separately), but also by aiding in squeezing out information about a third protein conformation that forms during the denaturation of α-CT.