Glossoscolex paulistus hemoglobin (HbGp) has a molecular mass of 3.6 MDa, determined by analytical ultracentrifugation (AUC). HbGp presents high oligomeric stability, resistance to oxidation and affinity to oxygen binding. The quaternary structure of this macromolecule consists of 144 globin chains, and 36 additional chains, lacking the heme group, named linkers, organized in a double-layered hexagonal structure. This class of proteins presents a high potential in biomedical applications as a blood substitute. Thus, the annelid hemoglobins are studied by several research groups in the world. AUC studies have shown that HbGp dissociates, under several conditions, such as, alkaline pH, presence of surfactants and urea, into smaller subunits, the dodecamer, the tetramer, the trimer and the monomer d. Recent studies have shown that the HbGp-surfactant interactions induce various processes, such as, protein aggregation, oligomeric dissociation and denaturation. On the other hand, the oligomeric stability is strongly associated to the iron oxidation state, and the specific ligand coordinated to the metal center. Thus, the focus of this post-doctoral project is to characterize the kinetic and thermodynamic processes of HbGp oligomeric dissociation and unfolding, in the presence of urea and guanidine hydrochloride. Additionally, the studies of thermal unfolding, in the presence of small concentrations of both denaturants, will be developed by several spectroscopic and hydrodynamic techniques. These studies will allow evaluating the dissociation mechanism, the stoichiometry of the subunits in the whole oligomeric structure, and the stability of the native protein under different conditions. The comparative study on the effect of two chaotropic agents with different physico-chemical characteristics, a neutral (urea) and a cationic (guanidine hydrochloride) ones, will enable the evaluation of the contribution of the electrostatic interaction in protein-denaturant system. Moreover, studies to characterize the main sites of protein-surfactant interactions in the monomer d, will be performed through crystallization and molecular modeling techniques of this subunit. The molecular modeling studies with the tetramer abcd, trimer abc, monomer d, and dodecamer (abcd)3, will be performed to characterize the interactions between the surfactants and these subunits, aiming to advance in the understanding of the interactions between these subunits in the HbGp oligomeric structure. These studies will be developed using crystallization and molecular modeling tools, as well as, spectroscopic and hydrodynamic techniques, such as, optical absorption, circular dichroism, dynamic light scattering (DLS), electron paramagnetic resonance (EPR) and AUC.
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