Structural Flexibility and Disassembly Kinetics of Single Ferritins Using Optical Nanotweezers

Abstract

Ferritin, a spherical protein shell assembled from 24 subunits, functions as an efficient iron storage and release system through its channels. The influence of chemicals on ferritin's conformation, dynamics, and stability remains a debated subject essential for understanding the origins of iron-related diseases in living organisms, including humans. Ascorbic Acid (Vitamin C) is one of the major elements in the human body, recognised for its association with iron-related diseases. However, its specific influence on single proteins is barely explored. In this paper, by employing optical nanotweezers using double nanohole (DNH) structures, we examined the effect of different concentrations of ascorbic acid on ferritin's conformational dynamics. The dynamics of ferritin increased as the ascorbic acid concentration approaches saturation kinetics within the ferritin core. Once the acidic conditions reach pH 2, ferritin displayed unstable fluctuations, and eventually underwent a stepwise disassembly into fragments, with each step corresponding to a transmission level in the trapping signal. We discovered four critical fragments during its disassembly pathway, which are 22-mer, 12-mer, tetramer (4-mer), and dimer (2-mer) subunits. This work further tracked the kinetics of single-molecule disassembly, demonstrating that ferritin experiences a cooperative disassembly procedure. Understanding the impact of chemicals on ferritin holds importance for disease-related implications, potential bio-applications, and their role in iron metabolism, medical treatments, and drug development.