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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 . . . . . . . . . . . . . . . . 407 409 411 412 413 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 . . . . . . . . . . . . 414 416 416 418 419 419 420 421 421 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406 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. 407 408 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- 409 410 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 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