Optimization of detergents in solubilization and reconstitution of Aquaporin Z: A structural approach

Abstract

Background: The exceptional capacities of aquaporins in terms of water permeation and selectivity have made them an interesting system for membrane applications. Despite the multiple attempts for immobilizing the aquaporins over a porous substrate, there is a lack of studies related to the purification and reconstitution steps, principally associated with the use of detergents in solubilization and destabilization steps. This study analyzed the effect of detergents in Aquaporin Z solubilization, considering the purity and structural homogeneity of the protein.

Methods: The extraction process was optimized by the addition of detergent at the sonication step, which enabled the omission of the ultracentrifugation and resuspension steps. Two detergents, Triton X-100, and octyl-glucoside were also evaluated. Destabilization mediated by detergents was used as reconstitution method. Saturation and solubilization points were defined by detergent concentration and both, liposomes and proteoliposomes, were analyzed by size distribution and permeability assays. Detergent removal with Bio-beads was also analyzed.

Results: Octyl glucoside ensures structural stability and homogeneity of Aquaporin Z. However, high concentrations of detergents induce the presence of defects in proteoliposomes. While saturated liposomes create homogeneous and functional structures, solubilized liposomes get affected by a reassembly process, creating vesicle defects with anomalous permeability profiles.

Conclusions: Detergent concentration affects the structural conformation of proteoliposomes in the reconstitution process.

General significance: Since the destabilization process is dependent on vesicle, detergent, and buffer composition, optimization of this process should be mandatory for further studies. All these considerations will allow achieving the potential of Aquaporins and any other integral membrane protein in their applications for industrial purposes.

Keywords: Aquaporin; Destabilization mediated by detergents; Detergent solubilization; Protein quality control; Protein reconstitution.