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405
15
Self-assembling Protein
Cage Systems and
Applications in
Nanotechnology
Prof. Trevor Douglas1, Mark Allen2, Prof. Mark Young3
1
Department of Chemistry and Biochemistry, Montana State University, Bozeman,
MT 59717; Tel.: ‡ 1-406-9946566; Fax: ‡ 1-406-9945407;
E-mail: [email protected]
2
Department of Chemistry and Biochemistry, Montana State University, Bozeman,
MT 59717; Tel.: ‡ 1-406-9946855; Fax: ‡ 1-406-9945407;
E-mail: [email protected]
3
Department of Plant Sciences, Montana State University, Bozeman, MT 59717;
Tel.: ‡ 1-406-9945158; Fax: ‡ 1-406-9947600; E-mail: [email protected]
1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
406
2
Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
407
3
3.1
3.2
3.3
3.4
Ferritin . . . . . . . . . . . . . . . . . . . . . . . . . .
Using Ferritin for Nanoparticle Synthesis . . . . . .
Modification of the Outer Surface of Ferritin . . . .
Assembly of Ferritin into Two-dimensional Arrays
Use of Ferritin as a Photocatalyst . . . . . . . . . . .
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409
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412
413
4
Ferritin-like Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
413
5
5.1
5.1.1
5.1.2
5.2
5.2.1
5.3
5.4
5.5
Viruses as Protein Cages . . . . . . . . . . . . .
Cowpea Chlorotic Mottle Virus . . . . . . . . .
Mineralization of CCMV . . . . . . . . . . . . .
Encapsulation Based on Structural Transitions
Cowpea Mosaic Virus . . . . . . . . . . . . . . .
CPMV as Template for Surface Modification .
Norwalk Virus . . . . . . . . . . . . . . . . . . .
Virus-like Protein Cages . . . . . . . . . . . . .
Viruses for Gene Delivery . . . . . . . . . . . .
414
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421
421
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15 Self-assembling Protein Cage Systems and Applications in Nanotechnology
6
Outlook and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
423
7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
424
Glu
His
kDa
Dps
FLP
CCMV
CPMV
NV
glutamic acid
histidine
kilodalton
DNA-binding protein from starved cells
ferritin-like protein
cowpea chlorotic mottle virus
cowpea mosaic virus
Norwalk virus
1
Introduction
In both synthetic and natural reaction
systems, the ability to create well-defined
reaction containers is critical for maintaining a physical separation between the
reaction and the surroundings. In complex
systems such as cells, physical boundaries
are created that define and separate the
inside of the cell from the surrounding
environment. This is similar to the way a
synthetic chemist uses a beaker to contain a
reaction and keep it isolated from the
surrounding environment. Nature has provided a collection of unique size-constrained
reaction environments based on lipid or
other amphiphilic assemblies. These are
often collections of many thousands of
individual molecules, and the vesicle or
micellar systems exhibit associated distributions in aggregate size. In contrast, there are
many examples of proteins that adopt cagelike architectures, derived from discrete
numbers of subunits, having a distinct
interior cavity. In all of these protein cages
the constrained volume is separated, from
the outside, by a porous protein shell usually
2 ± 5 nm thick. These protein cages are giant
molecules and as such have narrow particlesize distributions and can act as unique size-
constrained reaction vessels with potential
opportunities for nanotechnology that are
beginning to be explored. In this contribution, we will outline some examples of
protein cages being used in the growing
field of nanotechnology.
There are two major classes of protein cage
structures that have been used as building
blocks and templates for nanomaterials.
These are the protein cages derived from
viruses and the ferritin-like protein cages.
The ferritin-like proteins represent a fairly
narrow range of structural architectures
(Bozzi et al., 1997; Harrison and Arosio,
1996), while the virus-derived protein cages
exhibit a very wide range of sizes and
morphologies (Reddy et al., 2001). Other
examples of protein cages include lumazine
synthase (Schott et al., 1990) and the chaperones and heat shock proteins that also
form closed shell cage-like architectures
(Kagawa et al., 1995; Koeck et al., 1998;
Trent, 1996; Trent et al., 1997).
The approach to utilizing these cage-like
architectures for nanotechnology has exploited two spatially distinct interfaces
presented by the cages: the exterior and
the interior surfaces. In both the virus and
the ferritin-like examples, these interfaces
have been used to affect specific reactivities
different from the native functionality.
3 Ferritin
Extensive modification of these structures,
using either chemical modification or a
genetic-based approach, has shown the
potential plasticity of these structures as
molecular templates with some similarities
to the core-shell structures of dendrimers.
surface of the viral proteins provides a
diversity of chemical functionality for sitespecific modification to generate organic
materials as well as inorganic±organic composites ( Wang et al., 2002a,b,c).
2
3
Historical Outline
Ferritin
The iron storage protein ferritin was first
described in 1937 as a protein isolated from
horse spleen containing about 20% iron
(Laufberger, 1937). Later it was suggested
that an iron oxide colloid might ™ºform a
center surrounded by apoferritin molecules,
reminding on of the very open structures of
the zeolites∫. (Granick and Michaelis, 1942).
The first crystal structure of ferritin was
reported in 1991 (Lawson et al., 1991) where
the cage-like architecture of the protein
micelle was immediately clear. There are
now approximately 30 structures of ferritin
and ferritin-like proteins. The material
characteristics of the ferritin aggregate were
recognized early on (Granick and Michaelis,
1942), but it was not until 1991 that Mann
et al. showed that the protein structure could
be used as a synthetic reaction environment
for nanomaterials synthesis (Mann and
Meldrum, 1991). This approach was exploited in the synthesis of a number of proteinconstrained nanomaterials (Douglas, 1996),
and the use of ferritin as a synthetic reaction
vessel is ongoing (Douglas and Stark, 2000).
The cage-like properties of viral capsids were
recognized, and in 1998 the first use of
viruses as nanomaterials was presented
(Douglas and Young, 1998). Since then,
other viral capsids have been used, and the
wild-type virus has been substantially modified to impart new chemical functionality
(Douglas and Young, 1999; Douglas et al.,
2002). It was also recognized that the outer
Ferritins represent a family of proteins
rather than a specific single protein and
are found almost ubiquitously in biological
systems (Chasteen and Harrision, 1999;
Harrison and Arosio, 1996). Although there
are differences between prokaryotic and
eukaryotic ferritins, their overall similarities
are more important than their differences.
Ferritins are large multi-subunit proteins (24
subunits) that self-assemble to form a cagelike architecture (Figure 1a) with a central
cavity in which a hydrated ferric oxide (or
phosphate) is mineralized. Variations in
subunit composition do not seem to significantly influence the structure and function
of these proteins.
Mammalian ferritin has been found to
consist of mixtures of two different subunit
types known as H (heavy) and L (light)
chain, designations based on their molecular
weights (21 kDa and 20 kDa, respectively).
While there are differences between the
subunit amino acid sequences, vertebrate
ferritins show a high degree of identity (90%
between human, chicken, and rat H chain)
(Ford et al., 1984). Ratios of H to L chain
subunits in ferritin molecules are found to
vary between organisms as well as between
tissues within a particular organism (Grossman et al., 1992). Bacterioferritins (BFr)
resemble eukaryotic ferritins in many aspects except for amino acid sequence, where
only approximately 25% sequence identity is
found.
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15 Self-assembling Protein Cage Systems and Applications in Nanotechnology
Fig. 1 (a) Ribbon diagram of the 24-subunit ferritin protein cage (pbd file: 1ier) (b) Subunit of ferritin
comprising 5 a helical segments.
The two subunit types in mammalian
ferritins, H and L, are structurally similar.
Each subunit comprises a bundle of 4 ahelices (Figure 1b). This 4-helix bundle is
capped by a fifth helical section that lies at
roughly 608 to the bundle axis. In the
quaternary structure of ferritin, subunits
are aligned in 12 sets of antiparallel pairs
giving rise to a roughly rhombic dodecahedron shape. Subunit interactions lead to a
packed shell with three-fold and four-fold
symmetry axes where small channels intersect the protein as shown in Figure 1. From
X-ray structural determination (Harrison
et al., 1991), these channels are about 3 ä
in diameter. The three-fold channels are
hydrophilic in nature, having aspartate and
glutamate residues protruding into the
channel. On the other hand, the four-fold
channels are surrounded by four helices
(each from a different subunit) and the
channel is largely hydrophobic.
Calculations to determine the electrostatic
potential of the human H-chain homopolymer (HuHF ), reveal novel aspects of the
protein (Douglas and Ripoll, 1998). Some of
the calculated charge density correlates well
with regions previously identified as active
sites in the protein. The three-fold channels,
the putative ferroxidase sites, and the
nucleation sites all show expectedly negative
values of the electrostatic potential. However, the outer entrance to the three-fold
channels is surrounded by regions of
positive potential, creating an electrostatic
field directed toward the interior cavity. This
electrostatic gradient provides a guidance
mechanism for cations entering the protein
cavity (Figure 2), indicating the three-fold
channel as the major entrance to the protein
(Chasteen and Harrision, 1999; Harrison
and Arosio, 1996).
The mineralization of ferrihydrite within
ferritin is a multi-step process involving
Fe(II ) oxidation, hydrolysis, nucleation, and
crystal growth. Fe(II ) ions pass into the
cavity of the protein through the three-fold
channels. Metal-binding sites on H-chain
subunits (involving the residues Glu 27, Tyr
34, Glu 61, Glu 62, His 65, Glu 107, and Glu
141), but absent from L-chain subunits, have
been identified as the ferroxidase center
3 Ferritin
Fig. 3 Putative ferroxidase site in human H ferritin
showing the formation of a diferric-m-peroxo intermediate.
Calculated gradients in the electrostatic
potential at the 3-fold axis of human H ferritin.
Fig. 2
responsible for Fe(II ) oxidation (Lawson
et al., 1991). Oxygen binding to Fe(II ) at this
site undergoes a 2-electron reduction to form
a diferric-m-peroxo intermediate (Hwang
et al., 2000; Pereira et al., 1998), which
subsequently decomposes to form H2O2
and a ferric, Fe(III), species (Figure 3).
While the ferroxidase sites are present only
in a subset of the ferritin subunits, mineral
nucleation sites comprising clusters of
carboxylic acid residues are present in all
ferritin subunits. In human ferritin glutamic acid, residues (Glu 61, Glu 64, Glu 67)
on the inner surface are believed to aid the
nucleation process by aggregating ions at the
protein interface (Chasteen and Harrison,
1999; Harrison and Arosio, 1996; Mann
et al., 1992). Mutant proteins with key
residues deleted, from both the ferroxidase
and nucleation sites, showed complete
inhibition of mineralization (Lawson et al.,
1989).
The stoichiometry of Fe(II ) oxidation by
O2 is dependent on the subunit composition
(Xu and Chasteen, 1991; Yang et al., 1998).
Thus, reaction at the H-chain subunits
having the ferroxidase center follows the
stoichiometry outlined in Equation 1, in
which O2 acts as a 2-electron oxidant and
H2O2 and ferrihydrite are the products. In
the presence of L-chain subunit, there is no
ferroxidase reaction, and O2 acts as a 4electron oxidant and the stoichiometry
follows that of Equation 2. Furthermore,
the mechanistic process of building up the
core appears to have two components (Sun
and Chasteen, 1992). With low levels of Fe
loading, the ferroxidase activity of the
protein dominates according to Equation 1,
but at higher loading levels of Fe, the
growing mineral surface catalyses the oxidation reaction according to the stoichiometry of Equation 2.
2 Fe2‡ ‡ 2 H‡ ‡ O2 ! 2 Fe3‡ ‡ H2O2
(1)
4 Fe2‡ ‡ 4 H‡ ‡ O2 ! 4 Fe3‡ ‡ 2 H2O
(2)
The generation of Fe3‡ results in the
hydrolysis reaction to form a precipitate of
the
ferric
oxyhydroxide
ferrihydrite
(Fe(O )OH ) according to Equation 3.
‡
Fe3‡
…aq† ! (Fe(O )OH )(s) ‡ 3 H
(3)
3.1
Using Ferritin for Nanoparticle Synthesis
De-mineralized ferritin (apoferritin) is a
hollow, spherical protein shell homogene-
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15 Self-assembling Protein Cage Systems and Applications in Nanotechnology
ously dispersed in aqueous media. For
mineralization to occur within the confines
of the protein, rather than in the bulk
solution, the system needs to be chemically
biased so that reaction inside the protein
shell is favored over reaction outside the
protein. There are clear instances in the case
of ferritin where protein-assisted mineralization occurs, making mineralization inside
the protein cage faster than bulk precipitation. While this discrimination between
inside and outside is crucial to the effective
functioning of the protein in vivo, it is also
central to synthetic approaches exploited in
the formation of nanophase materials within
the protein shell of ferritin (Douglas, 1996).
The native mineralization of ferrihydrite
inside ferritin represents a process with the
highest degree of control, where the cooperative effects between the active sites (metallo-oxidase and nucleation) and the quaternary structure of the protein result in highly
spatially controlled mineralization that occurs as a series of separate events: oxidation,
hydrolysis, crystal nucleation, and crystal
growth. This process is driven by spatial
control of the supersaturation. Thus, soluble
Fe(II ) is oxidized to the insoluble Fe(III )
inside the protein cage (Figure 4). This
process can be controlled by the presence
of the ferroxidase site, but even in its
absence, a high degree of spatial selectivity
for the oxidation and mineralization process
occurs. In synthetic reactions, use of metal
ions other than Fe(II ), which are not known
to be substrates for the ferroxidase reaction,
result in the formation of cores exclusively
within the protein cage. Thus, Mn(II )
undergoes air oxidation and mineralization
to form Mn(O )OH and Mn3O4 within the
cage (Mackle et al., 1993; Mann and Meldrum, 1991; Meldrum et al., 1995). Co(II )
also undergoes oxidation, with H2O2 as
oxidant, to form a Co(O )OH mineral within
the cage (Douglas and Stark, 2000). A model
has been proposed in which the high charge
density of the nucleation sites on the interior
surface allows us to rationalize the spatial
selectivity of mineralization of these oxidative mineralization reactions (Douglas and
Stark, 2000). The Gouy-Chapman theory
predicts that a surface with a net charge will
aggregate counter ions at the interface and
that the concentration of these counter-ions
decreases exponentially with distance from
the interface until bulk concentration is
achieved. This suggests that the nucleation
sites will aggregate transition metal ions at
the protein interface, thereby facilitating
both the oxidation and subsequent aggregation/mineralization.
In the absence of all site-directed control,
mineralization is initiated randomly
throughout the system with extensive bulk
precipitation as well as some adventitious
mineralization within the protein shell. This
results in the formation of discrete mineral
cores surrounded by the protein shell in
addition to bulk precipitation. Using this
approach for nanoscale synthesis, the protein can be separated from the bulk precipitation after the reaction is complete,
Fig. 4 Transmission electron micrograph of the
iron oxide core within the ferritin protein cage.
3 Ferritin
although yield in these reactions is expected
to be extremely low. This approach has been
utilized in the formation of a uranyl oxyphosphate material encapsulated within the
protein cage (Hainfield, 1992; Mann and
Meldrum, 1991). This ionic crystallization
occurs within the protein cage of ferritin, but
because of the high levels of supersaturation
in the bulk medium, there is also significant
non-specific bulk precipitation.
Another approach to mineral formation
inside this spatially defined protein utilizes a
process whereby one mineral nanoparticle is
converted into another within the confines
of the organic matrix of the protein. The
process relies on a preexisting core that can
be transformed by an appropriate chemical
reaction. The core size of the new mineral in
this mechanism is controlled (or limited) by
the size of the preexisting core. The protein
matrix serves to contain the minerals within
its volume and possibly also plays some part
in the nucleation of the new mineral phase.
As an illustration of this, the ferrihydrite core
of ferritin can be converted into an iron
sulfide nanoparticle by exposure of the
mineralized particle to H2S (Douglas et al.,
1995; Mann and Meldrum, 1991). When this
is done at elevated pH, conversion of the
oxide to the sulfide occurs only on the
surface of the nanoparticle (Mann and
Meldrum, 1991). However, when the reaction is done at low pH, a process of
dissolution±reprecipitation occurs, whereby
the entire ferric oxide nanoparticle is converted into the corresponding iron sulfide
(Douglas et al., 1995).
The high charge density of the inner
surface and specific binding of Cd2‡ ions
also have been utilized for the synthesis of
protein-encapsulated CdS nanoparticles
( Wong and Mann, 1996). A CdS core was
synthesized by repeated treatments of apoferritin with substoichiometric amounts of
Cd2‡, followed by exposure to S2±. In this way,
the bulk concentration of free Cd2‡ was kept
exceedingly low, preventing bulk precipitation of CdS upon exposure to S2±.
Recently, a novel approach to using ferritin
as a cage-like host for molecular entrapment
has been demonstrated (Aime et al., 2002).
The ferritin cage was disassembled under
low pH conditions and subsequently reassembled at near neutral pH in the presence of a
Gd-chelate complex, GdDOTP (H8DOTP ˆ
1,4,7,10-tetrakis(methylenephosphonic acid)1,4,7,10-tetraazacyclododecane). This resulted in the entrapment of some of the Gdchelate complex within the protein cage.
This material has been shown to exhibit
extremely high NMR proton relaxivities,
making it a promising candidate for applications in magnetic resonance imaging.
The difference between mineralization
within the protein and bulk precipitation is
essentially a kinetic one, as illustrated by the
fact that mineral cores are often kinetically
stabilized mineral phases, different from
those formed in protein-free control experiments.
3.2
Modification of the Outer Surface of Ferritin
The outside surface of ferritin provides a rich
synthetic template for chemical modification. Carbodiimide activation of surfaceexposed carboxylates (glutamate and aspartate) allows coupling to primary amines
(Figure 5). In this way, carbodiimide-activated ferritin has been reacted with a diamine to
change surface acid groups to terminal
amines, thereby altering the overall charge
of the protein (Danon et al., 1972). The
protein has a significantly higher isoelectric
point and has been termed ™cationic ferritin∫.
Using essentially the same chemistry, alkylated derivatives of ferritin have been prepared by coupling of long-chain primary
amines to surface carboxylic acid residues
411