The contrasting effect of macromolecular crowding and confinement on fibril formation of globular protein: Underlying cause of proteopathies

Abstract

Protein molecules need to maintain a proper native conformation to perform encoded role in sustaining life. Naturally, the cellular milieu is densely crowded by different macromolecules, which can alter the secondary structure of protein eventually leading to protein aggregation which have been reported liable to many proteopathies. Hemoglobin (Hb), being an important alpha helical dominant globular protein, can serve as an excellent model to carryout structural transition studies. The aggregation profiling of Hb was done using turbidometric, thioflavin T, and Congo red assays. The secondary structural transition was confirmed by circular dichroism. Microscopic analysis using scanning electron microscope (SEM) show morphology of ordered Hb aggregates of protofibrils and fibril types. Generation of ROS (reactive oxygen species), genotoxic and cytotoxic potentials of Hb aggregates affirmed by DCFH-DA (2′,7′-dichlorofluorescin diacetate), DAPI (4′,6-diamidino-2-phenylindole) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay respectively. Our findings suggested that macromolecular crowding promotes Hb aggregation and the morphology of aggregates goes with the type of crowding agent used (polyethylene glycol 4000 (P4), 6000 (P6) and dextran 70 (D70)). Moreover, Hb aggregates causes redox perturbation and cytotoxicity in isolated human peripheral blood cells (PBMCs) which concludes the toxic potential and the gained toxic properties of Hb aggregates correlating to in vivo clinical conditions involved in various proteopathies including neurodegenerative disorders.