Detection and resolution of intermediate species in protein folding processes using fluorescence and circular...

Thermally induced protein unfolding/folding processes have been studied on alpha-lactalbumin and alpha-apolactalbumin. Experiments monitored by fluorescence and circular dichroism spectroscopic techniques on alpha-apolactalbumin showed the formation of an intermediate species, whereas in the case of alpha-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.