Detection and Resolution of Intermediate Species in Protein Folding Processes Using Fluorescence and Circular Dichroism Spectroscopies and Multivariate Curve Resolution
Susana Navea, Anna de Juan, Romà Tauler
Thermally induced protein unfolding/folding processes have been studied on α-lactalbumin and α-apolactalbumin. Experiments monitored by fluorescence and circular dichroism spectroscopic techniques on α-apolactalbumin showed the formation of an intermediate species, whereas in the case of α-lactalbumin, this intermediate species was not detected. The presence and resolution of this intermediate species, its spectrum, and the evolution of all conformations during protein unfolding/folding processes were estimated using the multivariate curve resolution-alternating least-squares method. Elucidation of the nature and contribution of the different secondary structure motifs in each of the resolved protein conformations, including the intermediate, was also carried out. Multivariate resolution has shown to be an excellent tool for the complete characterization of all protein conformations involved in folding processes, including intermediate species that cannot be isolated by physical or chemical means. Indeed, it is in the determination and modeling of these intermediates that this chemometric approach outperforms in power and reliability previous methodologies based on simpler measurements and data treatments and fills the void linked to the elucidation and interpretation of complex mechanisms in protein folding processes.
Circular dichroism, Protein denaturation, Chemometrics, Protein folding, Secondary structure, Biochemistry,