The Protein Shells

A nucleic acid can never code for a single protein molecule that is big enough to enclose and protect it. Therefore, the protein shell of viruses is built up from many copies of one or a few polypeptide chains. The simplest viruses have just one type of capsid polypeptide chain, which forms either a rod-shaped or a roughly spherical shell around the nucleic acid. The simplest such viruses whose three-dimensional structures are known are plant and insect viruses the rod-shaped tobacco mosaic virus, the spherical satellite tobacco necrosis virus, tomato bushy stunt virus, southern bean mosaic vims. 

The protein shells of spherical viruses have icosahedral symmetry... 

The asymmetric unit of an icosahedron can contain one or several polypeptide chains. The protein shell of a spherical virus with icosahedral symmetry... 

The cores of individual mammalian ferritin molecules are clearly visible by transmission electron microscopy as well defined nanoparticle crystallites encapsulated within the protein shell which can attain a size close to that of the 8 nm interior diameter of the protein shell (Massover, 1993). This is illustrated in Figure 6.13 for a sample of human ferritin. The amount of iron in the core is variable, and can range from zero to a maximum of approximately 4500 atoms (Fischbach and Anderegg, 1965) this corresponds to the capacity of the internal cavity for Fe(III) as... 

The initial stages of iron incorporation requires the ferroxidase sites of the protein. Thereafter the inner surface of the protein shell provides a surface which supplies ligands that can partially coordinate iron but which leave some coordination spheres available for mineral phase anions, thereby enabling the biomineralization process to proceed, with formation of one or more polynuclear ferrihydrite crystallites. Iron is transferred from the ferroxidase sites to the core nucleation sites by the net reaction (Yang et ah, 1998) ... 

About a quarter of the total body iron is stored in macrophages and hepatocytes as a reserve, which can be readily mobilized for red blood cell formation (erythropoiesis). This storage iron is mostly in the form of ferritin, like bacterioferritin a 24-subunit protein in the form of a spherical protein shell enclosing a cavity within which up to 4500 atoms of iron can be stored, essentially as the mineral ferrihydrite. Despite the water insolubility of ferrihydrite, it is kept in a solution within the protein shell, such that one can easily prepare mammalian ferritin solutions that contain 1 M ferric iron (i.e. 56 mg/ml). Mammalian ferritins, unlike most bacterial and plant ferritins, have the particularity that they are heteropolymers, made up of two subunit types, H and L. Whereas H-subunits have a ferroxidase activity, catalysing the oxidation of two Fe2+ atoms to Fe3+, L-subunits appear to be involved in the nucleation of the mineral iron core once this has formed an initial critical mass, further iron oxidation and deposition in the biomineral takes place on the surface of the ferrihydrite crystallite itself (see a further discussion in Chapter 19). 

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... 

As we pointed out earlier, the H subunit catalyses the ferroxidase reaction, which occurs at all levels of iron loading, but decreases with increasing amounts of iron added (48-800 Fe/ protein). Reaction (19.8) catalysed by both FI- and L-chain ferritins, occurs largely at intermediate iron loadings of 100-500 Fe/protein. Once nucleation has taken place, the role of the protein is to maintain the growing ferrihydrite core within the confines of the protein shell, thus maintaining the insoluble ferric oxyhydroxide in a water-soluble form. 

Why mammalian ferritin cores contain ferrihydrite-like structures rather than some other mineral phase is less easy to understand, and presumably reflects the way in which the biomineral is built up within the interior of the protein shell together with the geometry of the presumed nucleation sites. The phosphate content in the intracellular milieu can readily be invoked to explain the amorphous nature of the iron core of bacterioferritins and plants. Indeed, when the iron cores of bacterioferritins are reconstituted in the absence of phosphate, they are found to be more highly ordered than their native counterparts, and give electron diffraction lines typical of the ferrihydrite structure. Recently it has been reported that the 12 subunit ferritin-like Dps protein (Figure 19.6), discussed in Chapter 8, forms a ferrihydrite-like mineral core, which would seem to imply that deposition of ferric oxyhydroxides within a hollow protein cavity (albeit smaller) leads to the production of this particular mineral form (Su et al., 2005 Kauko et al., 2006). 

Formation of ferritin involves assemblage of the protein subunits to form the apo-ferritin shell which is then filled with the phosphated ferrihydrite core. The mechanism by which ferritin is filled and the iron core built up, has been investigated intensively in vitro. The experiments usually involved incubating apoferritin (from horse spleen) with Fe salts in the presence of an oxidant such as molecular oxygen. They showed that ferritin could be reconstituted from apoferritin and a source of Fe both the iron and the oxygen enter the protein shell, whereupon oxidation of Fe is catalysed by the interior surface of the protein shell (Macara et al., 1972). 

It is thought that oxidation of Fe takes place at specific sites within the protein shell and is followed by inward migration and hydrolysis to form a stable core nu-... 

An alternative mechanism has been proposed by Schneider (1988) who considers that ferritin could be also filled via a transient, mononuclear Fe " species. This species is similar to Fe in size, but is more versatile in its interaction with the protein shell. Experiments have shown that as the pH of a system containing diferric-trans-ferrin and ferritin was lowered very slowly from 7.5 to 5.0, monomeric Fe was released from the transferrin and redeposited in the ferritin (Glaus, 1989). Calculations of the iron flux across the cell membrane and estimates of the rates of interaction of the mononuclear species with ferritin and with the cell mitochondria indicated that the steady state concentration of the mononuclear Fe species would be sufficiently low for this species alone to enter the protein shell and be deposited as the iron core. Uptake of this species by the protein shell is about fiftyfold faster than the rate of hydrolytic polymerization or even of the dimerization of Fe (tiy2 1 vs. 50 ms). This hypothesis suggests an interesting direction for further research. 

Ferritin can be converted in situ at 60°C to magnetoferritin by addition of Fe " ions (Meldrum et al. 1992). This process is an example of the use of the protein shell and other small volumes to synthesize nanominerals (Mann et al. 1993). 

Figure 16-3 Structure of the protein shell of ferritin (apoferritin). (A) Ribbon drawing of the 163-residue monomer. From Crichton.62 (B) Stereo drawing of a hexamer composed of three dimers. (C) A tetrad of four subunits drawn as a space-filling diagram and viewed down the four-fold axis from the exterior of the molecule. (D) A half molecule composed of 12 subunits inscribed within a truncated rhombic dodecahedron. B-D from Bourne et al.7i...

Ferritin has also been compared with iron-dextran by the EXAFS technique.1108 The apoferritin controls the deposition of the core. Reconstitution of ferritin under a range of conditions always gives the same structure, which is not the case in the absence.of apoferritin. There are metal-binding sites on the protein shell. There is evidence for the binding of iron to apoferritin, probably by carboxyl groups, but there is little detailed information on these sites.1098 On the other hand, other metal ions inhibit the formation of ferritin and may do this by binding at or close to the iron sites. Of most significance appear to be results on Tb3+, Zn2+ and V02+,... 

The mechanism of deposition and mobilization of iron in ferritin has been much discussed.1106,1112,1113 Ions and neutral molecules can all pass through the protein shell, due probably to the presence of entry channels of different character. The Cd2+ ions found in the hydrophilic channel in the crystal structure could be metal ions entering the shell. 

For the detection of viruses different SERS approaches are reported. One possibility is the direct detection of the DNA or the protein shell. Additionally, the presence of the DNA can also be detected indirectly via marker molecules. The third possibility is the detection via antibodies [28],... 

CPMV particles have an icosahedral symmetry with a diameter of approximately 28 nm (Figure 9.2), the protein shell of the capsid is about 3.9nm thick [72], The structure of CPMV is known to near-atomic resolution (Figure 9.3) [73], The virions are formed by 60 copies of two different types of coat proteins, the small (S) subunit and the large (L) subunit. The S subunit (213 amino acids) folds into one jelly roll P-sandwich, and the L subunit (374 amino acids) folds into two jelly roll P-sandwich domains. The three domains form the asymmetric unit and are arranged in a similar surface lattice to T = 3 viruses, except they have different polypeptide sequences therefore the particle structure is described as a pseudo T = 3 or P = 3 symmetry [74]. 

---