Folding channel

Ferritin is a globular iron-storage protein that stores iron as FeJ+. To leave the ferritin, Fe3+ must first be reduced to Fe2+. Ferritin has two types of channels through which the Fe"+ could leave a three-fold channel and a four-fold channel. The three-fold channel is lined with the amino acids aspartate (Asp) and glutamate (Glu) and the four-fold channel is lined with the amino acid leucine (Leu). Through which channel is the Fe + more likely to leave the ferritin protein Explain your answer. 

It is unfortunately the case that when we incubate apoferritin with a certain number of iron atoms (for example as ferrous ammonium sulfate), the product, after elimination of non-protein-bound iron, does not have a homogeneous distribution of iron molecules which were able to (i) take up iron rapidly through the three fold channels, (ii) quickly transfer it and form a diiron centre on a ferroxidase site, and (iii) to transfer the iron inward to a nucleation site, where (iv) it will begin to catalyse iron oxidation on the surface of the growing crystallite, will accumulate iron much more rapidly, and in much greater amounts than molecules in which steps (i), (ii) and (iii) are slower, for whatever reasons (perhaps most importantly subunit composition, and the disposition of subunits of the two types H and L, one with regard to the other). This polydispersity makes the analysis of the process of iron uptake extremely difficult. 

Access of iron to the interior of the protein could be through channels, which traverse the shell along the three- and four-fold axes of symmetry of the protein. The three-fold channels are predominantly hydrophilic, with three glutamate and three aspartate residues at each end of the funnel-shaped channel. In contrast, the four-fold channels are essentially lined with hydrophobic residues. 

Iron incorporation into mammalian ferritins is thought to involve the following steps (Crichton, 2001) (1) Uptake of Fe2+ into the protein shell, most probably through the hydrophilic three fold channels. (2) Oxidation of ferrous iron by the dinuclear ferroxidase... 

We assume that all substances involved in ferritin iron deposition (Fe2+, Fe3+, 02) need to gain access to the interior of the apoferritin protein shell. The most likely pathway is via the three-fold channels, which would involve passing through the 12 A long channel, and then traverse a further distance of 8 A along a hydrophilic pathway from the inside of the... 

Low Fe/Proteln (Fe <10-12/Molecule). Ferritin protein coats have multiple (8-12) binding sites for a variety of metals. Including Fe(II), Fe(IIl), V(IV), Mn(II), Tb(III), Cd(II), Zn(II), and Cu(II) (e.g., 5,34-36, and reviewed In Ref. 37). At least some of the metals bind at the three-fold channels. The location of the nucleatlon sites Is presently unknown. However, If the three-fold channels are the nucleatlon sites for core formation, core growth could block the channels, thus Inhibiting further accretion INSIDE the protein coat and could lead to the addition of Fe OUTSIDE the protein coat. Such an effect would obviate the sequestering function of the protein. Three forms of Fe have been observed bound to ferritin protein coats (apoferrltln) mononuclear, dlnuclear, and multlnuclear clusters. 

Griffiths, S.K., Nilson, R.H., Design and analysis of folded channels for chip-based separations. Anal. Chem. 2002, 74(13), 2960-2967. 

Fig 8, Stereo view of the region around a four-fold channel in human rHF viewed from inside the cavity. The channel is surrounded by four histidines (cavity side I and eight leucines. 

Fio 9. Stereo diagram of the three-fold channel region of human rHF viewed from outside the molecule. Only the narrow end of the channel, toward the cavity, is shown. In the center of the channel there is a large peak of electron density presumed to represent a Ca - ion (marked here with a star). It has six oxy ligands supplied by three glutamates and three waters (hydrogen bonded to aspartates) in octahedral geometry. 

An alternative route through the shell has been found in rHF. There appears to be a narrow passage (or one-fold channel) in the subunit leading directly to the ferroxidase center 94, 115). This would give a pathway of about 12 A to this site compared with over 50 A via the three-fold channels. However, the route or routes by which Fe(II) reaches the ferroxidase center remains uncertain. In L ferritins, in which the one-fold channel is blocked, entry into the cavity may be via the three-fold channels. 

Fig. 13. Stereo view of the inner, narrow part of the three-fold channel region of a variant of human rHF in which Asp 131 has been replaced by His. The large peak of electron density seen in the center three-fold channels of wild type rHF (Fig. 9) is absent. Instead, a low-density peak that may represent a water molecule H-bonded to three His 131 molecules is found and is shown here. Note that because of differences in ligand preferences, the absence of a Ca + site in these channels does not necessarily mean that Fe + cannot bind here.

An alternative suggestion has been made that Fe(III) clustering starts in the three-fold channels and that the core grows from one of these positions (114). However, this can be ruled out, because core formation proceeds in variants in which the channel aspartates and glutamates have been changed (109). 

Fe(II) must then move from the 12 A long channel, and traverse a further distance of about 8 A along a hydrophilic pathway from the inner side of the three-fold channel to the ferroxidase site, and a putative pathway for Fe(II) is shown in Figure 19.6. The diiron ferroxidase centre is located in the central region of the four-helix subunit bundle and the coordination geometry of the ferroxidase centre of human H-chain ferritin is shown in Figure 19.7. Detailed analysis of the ferroxidase reaction in H-chain ferritin has allowed the identification of a number of intermediates, which are illustrated in Figure 19.8. 

FIGURE 19.6 Internal view of the three-fold channel of ferritin (left panel). The right panel shows a putative Fe(II) pathway from the threefold channel to the ferroxidase centre of the protein. (From Bou Abdallah, 2010. Copyright 2010 with permission from Elsevier.)... 

Fig. 5. Scheme of a two-step binding mechanism between ICAM-1 and major group HRVs. The first step, on the left, is observed in the cryo-EM reconstructions of HRV-ICAM-1 fragments. On the right, the second (hypothesized) step involves a conformational change in the virus surface. The five-fold channel may open as both walls and floor of the canyon bind to domain Dl of ICAM-1. The pocket region is postulated as the hinge area, and needs to be empty in order to provide conformational flexibility. 

In EcFtna a relaxation spectrum c has been assigned mainly to mononuclear Fe(III) at site C, although the possibility that part of this spectrum is due to small clusters with an uneven number of Fe(III) atoms (e.g. trimers or pentamers) cannot be ruled out. In contrast, the Fe(III) relaxation spectrum observed in HuHF seems to be due mainly to iron ligated to aspartate and glutamates in the three-fold intersubunit channels [68]. Such iron would not be expected in EcFtna because carbox-ylates are absent from its three-fold channels [5]. 

Hisamoto, D., W.-C. Lee, J. Kedzierski, E. Anderson, H. Takeuchi, K. Asano, K, T.-J. King, J. Bokor, C. Hu. 1998. A folded-channel MOSFET for deep-sub-tenth micron era. Inlemational Electron Devices Meeting Technical Digest 1032-1034. 

Protein folding channels and kinetics of two-state folding... 

In consequence, the distribution Hj ucaiE, Q) can suitably be used to identify the folding channels, independently of temperature. This is more difficult with temperature-dependent canonical distributions F E, Q), which can, of course, be obtained from //mucaf. Q) by a simple reweighting procedure, Hm KaiE,Q)g E) tx i—E/ksT). Nonethe-... 

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