Nano-structured Hydrotrope-Caged Cytochrome c with Boosted Stability in Harsh Environments: A Molecular Insight

Abstract

Green and nanostructured catalytic media are vital in biocatalysis to attenuate the denaturation tendency of biocatalysts under severe reaction conditions. Hydrotropes with multifaceted physiochemical properties can be envisaged as promising systems for sustainable protein packaging. Herein, the suitability of adenosine-5'-triphosphate (ATP) and cholinium salicylate ([Cho][Sal]) ionic liquid (IL) to form nanostructures and to nanoconfine Cytochrome c (Cyt c) were disclosed envisioning enhancement of stability and activity under multiple stresses. Experimental and computational validations were undertaken to explain nanostructuring phenomenon of ATP and IL, structural organizations of nanoconfined Cyt c, and site-specific interactions that stabilize protein structure. Both, ATP and IL form nanostructures in aqueous media and caged Cyt c via multiple non-specific soft interactions. Remarkably, the engineered molecular nano-cages of ATP (5-10 mM), IL (300 mg/mL), and ATP+IL around Cyt c resulted in 9-72 folds higher peroxidase activity than native Cyt c with exceptionally high thermal tolerance (110 degrees C). The polar interactions mediated by hydrotropes with the cardiolipin binding site of Cyt c well corroborated with the increased peroxidase activity. Further, higher activity trends were observed in the presence of urea, GuHCl, and trypsin without any protein degradation. Specific binding of hydrotropes at highly mobile regions of Cyt c (Omega 40-54 residues) and enhanced H-bonding with Lys and Arg offered excellent stability under extreme conditions. Additionally, ATP effectively counteracted reactive oxygen species (ROS) induced denaturation of Cyt c, which was enhanced by [Sal] counterpart of IL. Overall, this study explored the robustness of nanostructured hydrotropes having a higher potential for protein packaging with improved stability and activity in extreme conditions. Thus, the present work brings out a novel strategy for real-time industrial biocatalysis to protect mitochondrial cells from ROS-instigated apoptosis.