Detection and Resolution of Intermediate Species in Protein Folding Processes Using Fluorescence and Circular Dichroism Spectroscopies and Multivariate Curve Resolution

Abstract

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.

Noise propagation and error estimations in multivariate curve resolution alternating least squares using resampling methods

“…See References [6,7,11,14] for explanations of the use of the ALS iterative optimization procedure. MCR-ALS has been extensively used to analyze spectroscopic data obtained in biochemical and biophysical process monitoring [3,4,19] and other data systems [1,2]. Concentration profiles C and pure spectra S T found using this constrained alternating least squares optimization algorithm may differ from the true ones because of the unresolved ambiguities under the set of applied constraints [6,33].…”

Mechanisms underlying the toxic effects of antimony species in human embryonic kidney cells (HEK-293) and their comparison with arsenic species

“…Conformational changes and aggregation mechanisms have been generally studied using synthetic samples or biological samples, but these samples were subjected to strong post-treatment steps that could induce structural changes in proteins; the techniques used included fluorescence spectroscopy, circular dichroism, light scattering, gel electrophoresis, NMR, X-ray diffraction and transmission electronic microscopy (Arnesano et al, 2009;Kelly et al, 2005;Navea et al, 2002;Rolinski et al, 2010). However, these techniques cannot be directly used for protein aggregation studies in living cells.…”

Biological and Medical Applications of Multivariate Curve Resolution Assisted Raman Spectroscopy

“…This leads to the extraction of physically meaningful spectra that represent pure chemical components without any prior information concerning the nature of the sample. 26 In fact, it has already been made use of in many spectroscopic techniques, which include LC/MS, [27][28][29] [39][40][41] It is only recently that Raman spectroscopists have become interested in employing MCR to disentangle dynamic changes of multiple bio-molecular components in complex samples, like cells and tissues. This review provides an overview of applications of MCR assisted Raman spectroscopy, and focuses on some selected papers that emphasize its recent advancements, particularly in biological and medical sciences.…”