Ferritin Assembly Revisited: A Time-Resolved Small-Angle X-ray Scattering Study

Sato, Daisuke; Ohtomo, Hideaki; Yamada, Yoshiteru; Hikima, Takaaki; Kurobe, Atsushi; Fujiwara, Kazuo; Ikeguchi, Masamichi

doi:10.1021/acs.biochem.5b01152

Cited by 48 publications

(51 citation statements)

References 21 publications

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“…This supports the preferential formation of heteropolymers over the homopolymers. It should be noted that the kinetics we observed by FRET and confirmed by PAGE are slower that those measured by small-angle X-ray scattering of the bacterial ferritin (9). The difference may be attributed to the different ferritin types and that SAXRS follows the subunit aggregation and cannot distinguish a well-formed shell from an aggregate made of incompletely folded and poorly stable subunits.…”

Section: Discussionmentioning

confidence: 55%

“…Both concluded that the reaction goes to completion in less than an hour, and that the subunit dimer is the first essential step in the assembly (16,32,17). More recently the self-assembly reaction of bacterial ferritin EcFtnA has been studied by Small Angle-X-ray scattering technique (9), that suggested the existence of intermediate oligomers during the reaction and that it was complete in minutes. All these approaches considered the assembly of one subunit type to form homopolymers, but one interesting property of ferritin is the capacity to form heteropolymers made of different proportions of two subunit types, namely the H-and L-chain for mammalian ferritins.…”

Section: Discussionmentioning

confidence: 99%

“…This process has been exploited to encapsulate in the cavity molecules of pharmacological and chemical interest (3,4,5). This reaction, although highly used has been analyzed in detail only in a few reports (6,7,8) and the kinetics of the reassembly have been studied in detail only for ferritins of bacterial origin by using X-ray scattering (9).…”

Section: Introductionmentioning

confidence: 99%

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Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

Carmona

Poli

Bertuzzi

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background: The high stability and strong self-assembly properties made ferritins the most used proteins for nanotechnological applications. Human ferritins are made of 24 subunits of the H-and Ltype that coassemble in an almost spherical nanocage 12 nm across, delimiting a large cavity. The mechanism and kinetics of ferritin self-assembly and why H/L heteropolymers formation is favored over the homopolymers remain unclarified. Methods: We used the Fluorescence Resonance Energy Transfer (FRET) by binding multiple donor or acceptor Alexa Fluor fluorophores on the outer surface of human H and L ferritins and then denaturing and reassembling them in different proportions and conditions. Results: The FRET-efficiency increase from <0.3 of the disassembled to >0.7 in the reassembled allowed to study the self-assembly kinetics. We found that their assembly was complete in about one hour, and that the initial rate of self-assembly of H/L heteropolymers was slightly faster than that of the H/H homopolymers. Then, by adding various proportions of unlabeled H or L-chains to the FRET system we found that the presence of the L-chains displaced the formation of H-H dimers more efficiently than that of the H-chains. Conclusion: Heterodimeric (H/L) subunit association is preferred during H/L heteropolymers formation. The H-chains arrange at distant positions on the heteropolymeric shell until they reach a number above eight, when they start to co-localize and the ferroxidase activity of the heteropolymer reaches a plateau. General significance: This favored formation of H/L heterodimers, which is the initial step in ferritin self-assembly, contributes to explain the preferred formation of H/L heteropolymers over the H or L homopolymers. Keywords

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Section: Discussionmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

Carmona

Poli

Bertuzzi

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…This self‐assembly reaction has been extensively used to encapsulate inside the cavity chemicals of biotechnological interest and recently it has been analyzed more in detail using FTN from Escherichia coli and time‐resolved small‐angle X‐ray scattering (TR‐SAXS) technique. The ferritin was disassembled at low pH and a jump to neutral pH values induced a fast assembly: the analysis of the kinetics indicated that the process involves as intermediates tetramers, hexamers, and dodecamers and also that an electrostatic repulsion between the subunits slows the reaction, which was facilitated and accelerated by high ion strength . A study on the kinetics of the assembly of human ferritins was done using FRET technology by labeling H chains with a fluorescent donor probe and both H and L chains with an acceptor probe.…”

Section: Ferritin Self‐assemblymentioning

confidence: 99%

Ferritin, cellular iron storage and regulation

2017

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Ferritin is considered the major iron storage protein which maintains a large iron core in its cavity and has ferroxidase activity. There are many types of ferritin particularly in prokaryotes that include the canonical 24-mer FTN molecules, the heme-containing BFR, the smaller 12-mer DPS and the newly recognized EncFtn of encapsulin that forms a very large iron storage compartment. Recent studies show that ferritin function is more dynamic than previous depicted and new mechanisms of ferritin iron recycling are emerging. They participate to the regulation of cellular iron homeostasis as those of ferritin biosynthesis, cooperating also with the iron-dependent mechanism of cellular iron secretion. Some of these basic processes are in common between unicellular and animal cells, and this review aims at discussing the findings on the connections between iron storage, cellular iron regulation and ferritin iron recycling that have been explored in unicellular organisms and in animals. © 2017 IUBMB Life, 69(6):414-422, 2017.

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“…This means that apoferritin is disassembled for acidic condition. Time-resolved SAXS measurements have been utilized to study disassembling and reassembling process upon the change in pH [4,5]. In Figure 1, the curve shows the result of the SAXS modeling by the scattering program GNOM [6].…”

Section: Nanoparticles With a Narrow Size Distributionmentioning

confidence: 99%

SAXS Evaluation of Size Distribution for Nanoparticles

Sakurai¹

2017

X-Ray Scattering

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Size distribution is an important structural aspect in order to rationalize relationship between structure and property of materials utilizing polydisperse nanoparticles. One may come to mind the use of dynamic light scattering (DLS) for the characterization of the size distribution of particles. However, only solution samples can be analyzed and even for those, the solution should be transparent or translucent because of using visible light. It is needless to say that solid samples are out of range. Furthermore, the size distribution only in the range of several tens of nanometers can be characterized, so DLS is useless for particles in the range of several nanometers. Therefore, the small-angle X-ray scattering (SAXS) technique is much superior when considering the determination of the size distribution in several nanometers length scale for opaque solutions and for solid specimens. Furthermore, the SAXS technique is applicable not only for the spherical particle but also for platelet (lamellar) and rod-like (cylindrical) particles. In this chapter, we focus on the form factor of a variety of nanostructures (spheres, prolates, core-shell spheres, core-shell cylinders and lamellae). Also getting started with a monodisperse distribution of the size of the nanostructure, to unimodal distribution with a narrow standard deviation or wide-spreading distribution and finally to the discrete distribution can be evaluated by the computational parameter fitting to the experimentally obtained SAXS profile. In particular, for systems forming complicated aggregations, this methodology is useful. Not only the size distribution of 'a bunch of grapes' but also the size distribution of all 'grains of grapes in the bunch' can be evaluated according to this methodology. This is very much contrasted to the case of the DLS technique by which only 'a bunch of grapes' is analyzed but 'grains of grapes in the bunch' cannot be. It is because the DLS technique in principle evaluates diffusion constants of particles and all of the grains in the same bunch of grapes diffuse as a whole. Thus, the methodology is important to highlight versatility and diversity in real materials, especially in soft matter, both in the liquid and in the solid states.

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Ferritin Assembly Revisited: A Time-Resolved Small-Angle X-ray Scattering Study

Cited by 48 publications

References 21 publications

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

Ferritin, cellular iron storage and regulation

SAXS Evaluation of Size Distribution for Nanoparticles

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