Structural and Biochemical Characterization of Aedes aegypti Hsp70 in Response to Redox Stress

Mosquitoes, as ectothermic organisms, exhibit rapid adaptation and reproductive capabilities but face various physiological and environmental stresses affecting their development, population dynamics, and feeding habits. The Aedes aegypti mosquito, a critical vector for Dengue, Zika, and Chikungunya viruses, poses a significant public health challenge globally. Heat shock proteins (Hsps), particularly the Hsp70 family of molecular chaperones, play a vital role in responding to environmental stress. This study focuses on the structural and functional aspects of Aedes aegypti Hsp70 chaperone (AaHsp70). AaHsp70 was cloned into a pET28a(+) plasmid for recombinant expression in Escherichia coli Origami2 (DE3) strain at 20 °C for 24 hours. The protein was purified through Ni²⁺ affinity and size exclusion chromatography. Structural analysis was performed using Circular Dichroism (CD), Tryptophan fluorescence, Size Exclusion Chromatography coupled to Multi-angle and Quasi-Elastic Light Scattering (SEC-MALS-QELS). Chaperone activity was assessed via light scattering (turbidity) at 320 nm using a fluorometer (Aminco Bowman Series 2) equipped with a thermoelectric sample temperature controller (Peltier system). ATPase activity was measured using the ATP/NADH coupled method. Spectroscopy data confirmed that AaHsp70 maintained secondary and tertiary structures characteristic of Hsp70 orthologs. Thermal-induced unfolding revealed at least two distinct transitions, with stability influenced by nucleotide presence. Hydrodynamic analysis showed AaHsp70 in a monomeric state with a slightly elongated shape. Functionally, AaHsp70 protected lysozyme and insulin from aggregation under stress caused by DTT in more than 60%, and the Hsp40/DNAJ family stimulates AaHsp70’s intrinsic ATPase activity. The results enrich the knowledge of the Hsp70 molecular chaperone and allow us to understand the peculiarities of these macromolecules.