Efeito de íons cálcio e magnésio na estabilidade térmica do domínio N-terminal da Hsp701A

Abstract

Proteins are important for the homeostasis of organisms. For this to happen, they need to exist in their proper structure state and correct cellular location. In this context, it is given the importance of molecular chaperones of the Hsp70 family, proteins which are responsible for the quality control mechanism of protein folding, transport of them to cellular compartments, prevention of the formation of protein aggregates, among others. Therefore, defects in Hsp70 proteins may be related to several human pathologies, such as neurodegenerative diseases, diabetes and even some types of cancer. An important example of a member of this protein family is Hsp701A. This protein has two domains, the NBD, which binds to the nucleotides ATP and ADP, and the PDB, which binds to the client protein. In the present study, experiments were carried out to verify the biophysical and biochemical characteristics of the recombinant NBD of Hsp701A in the presence of Ca2+ and Mg2+ ions and ATP and ADP nucleotides. For this, the protein in question was expressed by a recombinant bacterial system, purified by two chromatography steps and then analyzed. Thus, it was verified that Hsp701A_NBD was produced in its pure, monomeric, folded and catalytic active state. In this context, it was possible to verify that the presence of divalent ions did not cause a significant change in the secondary structural content of the domain. In addition, it was also noticed that ions and nucleotides cause suppression of fluorescence, indicating complex formation. Moreover, in the presence of Ca2+, there was a shift of fluorescence to a region of higher energy, an increase in the thermal stability of the protein and an increase in the catalytic activity of breaking down ATP into ADP and Pi. Furthermore, calcium acts on molecular chaperones of the Hsp70 family increasing the affinity of NBD for ADP, which results in a slower release of client proteins. Therefore, the biophysical, hydrodynamic and biochemical evaluations demonstrated that the Hsp701A_NBD domain was pure, monomeric, folded and catalytically active and that changes in the presence of Ca2+ were more pronounced.