Optical Monitoring of In Situ Iron Loading into Single, Native Ferritin Proteins

Abstract

Ferritin is a protein that stores and releases iron to prevent diseases associated with iron dysregulation in plants, animals, and bacteria. The conversion between iron-loaded holo-ferritin and empty apo-ferritin is an important process for iron regulation. To date, studies of ferritin have used either ensemble measurements to quantify the characteristics of a large number of proteins or singlemolecule approaches to interrogate labeled or modified proteins.Here we demonstrate the first real-time study of the dynamics of iron ion loading and biomineralization within a single, unlabeled ferritin protein. Using optical nanotweezers, we trapped single apo-and holoferritins indefinitely, distinguished one from the other, and monitored their structural dynamics in real time. The study presented here deepens the understanding of the iron uptake mechanism of ferritin proteins, which may lead to new therapeutics for iron-related diseases.

Solid-State Nanopore Distinguishes Ferritin and Apo-Ferritin with Identical Exteriors through Amplified Flexibility at Single-Molecule Level

“…Very recently, Yousefi et al reported their excellent work about monitoring the flexibility changes of ferritin as the continue iron loading to apo-ferritin with optical methods. 52 Our result is consistent with their conclusion. In order to verify the feasibility of this strategy, a smaller nanopore with a diameter of 7 nm is also prepared.…”

“…Specifically, the flexible apo-ferritin is more likely to be influenced by external forces than rigid ferritin, resulting in larger amplitude fluctuations. Very recently, Yousefi et al reported their excellent work about monitoring the flexibility changes of ferritin as the continue iron loading to apo-ferritin with optical methods . Our result is consistent with their conclusion.…”

Structural Flexibility and Disassembly Kinetics of Single Ferritins Using Optical Nanotweezers

“…As the transmitted light intensity correlates to the conformation of the trapped protein, the root-mean-square (RMS) of the optical signals reveal the fluctuation of protein conformations. 23 The RMS of Figure 2b demonstrates an increased dynamic of ferritin within high concentrations of ascorbic acid, arising from the opening and closing of the 3-fold channels due to the permeation of ascorbate and ferrous iron. The pro-oxidant behaviour of high concentrations of ascorbic acid promotes protein’s channel opening and induces increased amplitude of the transmission signal.…”

In Situ Observation of Chemically Induced Protein Denaturation at Solvated Interfaces

“…Figure a shows a 3.0 Å resolution structure of human ferritin proteins obtained using cryo-electron microscopy (protein data bank identifier: 5Y15 ). The globular structure of ferritin (molecular weight: ∼450 kDa) is composed of 24 subunits (H- and L-chains) with an inner cavity for iron storage known from previous studies. − Here, we used L-chain-rich ferritins isolated from the human liver with their ferrihydrite core (see the Supporting Information Section S1 for details on sample preparation and buffer conditions). The large-area AFM scan in Figure b shows single ferritin proteins randomly adsorbed at the graphene water interface.…”