Evolution of an oxygenic atmosphere required primordial life to accommodate the toxicity associated with reactive oxygen species. We have characterized an archaeal antioxidant from the hyperthermophilic acidophile Sulfolobus solfataricus. The amino acid sequence of this approximately 22-kDa protein shares little sequence similarity with proteins with known function. However, the protein shares high sequence similarity with hypothetical proteins in other archaeal and bacterial genomes. Nine of these hypothetical proteins form a monophyletic cluster within the broad superfamily of ferritin-like diiron-carboxylate proteins. Higher order structural predictions and image reconstructions indicate that the S. solfataricus protein is structurally related to a class of DNA-binding protein from starved cells (Dps). The recombinant protein self assembles into a hollow dodecameric protein cage having tetrahedral symmetry (SsDps). The outer shell diameter is approximately 10 nm, and the interior diameter is approximately 5 nm. Dps proteins have been shown to protect nucleic acids by physically shielding DNA against oxidative damage and by consuming constituents involved in Fenton chemistry. In vitro, the assembled archaeal protein efficiently uses H2O2 to oxidize Fe(II) to Fe(III) and stores the oxide as a mineral core on the interior surface of the protein cage. The ssdps gene is up-regulated in S. solfataricus cultures grown in iron-depleted media and upon H2O2 stress, but is not induced by other stresses. SsDps-mediated reduction of hydrogen peroxide and possible DNA-binding capabilities of this archaeal Dps protein are mechanisms by which S. solfataricus mitigates oxidative damage.

In multistep reactions, stability of intermediates is critical to the rate of product formation and a significant factor in generating kinetic traps. The capsid protein of cowpea chlorotic mottle virus (CCMV) can be induced to assemble into spherical particles of 30, 60, and 90 dimers. Based on examining assembly kinetics and reaction end points, we find that formation of uniform, ordered structures is not always a result of reactions that reach equilibrium. Equilibration or, alternatively, kinetic trapping can be identified by a straightforward analysis. Altering the assembly path of "spherical" particles is a means of controlling the distribution of products, which has broad applicability to self-assembly reactions.

The 24 subunit heat-shock protein from Methanococcus jannaschii has been genetically altered to display a dodecapeptide on the interior surface that has a strong binding affinity to the L1(0) phase of CoPt (CP_Hsp). Reaction of Co(II) and Pt(II) salts at 65 degrees C under reducing conditions results in the formation of a stable CoPt mineral encapsulated within the protein cage. Metallic particles commensurate in size with the interior dimensions of the protein cage (6.5 +/- 1.3 nm) have been imaged by transmission electron microscopy and are shown to be surrounded by the intact protein cage. Magnetic measurements performed on the encapsulated nanoparticles exhibit room-temperature hysteresis on the order of 150 G (1 G = 10(-4) T) prior to annealing and 610 G after annealing at 650 degrees C.

We have developed methods for producing viral-based protein cages in high yield that are amenable to genetic modification. Expression of the structural protein of Cowpea chlorotic mottle bromovirus (CCMV) using the yeast-based Pichia pastoris heterologous expression system resulted in the assembly of particles that were visibly indistinguishable from virus particles produced in the natural host. We have shown that a collection of non-infectious CCMV coat protein mutants expressed in the P. pastoris system assemble into viral protein cages with altered architectures and function. This provides an alternative to other heterologous expression systems for production of viral structural proteins in which expression has resulted in unassembled cages. Heterologous expression in P. pastoris further enhances the development of viral-based protein cages as biotemplates for nanotechnology and for future studies examining details of icosahedral virus assembly.

The complete genome sequences of two Sulfolobus spindle-shaped viruses (SSVs) from acidic hot springs in Kamchatka (Russia) and Yellowstone National Park (United States) have been determined. These nonlytic temperate viruses were isolated from hyperthermophilic Sulfolobus hosts, and both viruses share the spindle-shaped morphology characteristic of the Fuselloviridae family. These two genomes, in combination with the previously determined SSV1 genome from Japan and the SSV2 genome from Iceland, have allowed us to carry out a phylogenetic comparison of these geographically distributed hyperthermal viruses. Each virus contains a circular double-stranded DNA genome of approximately 15 kbp with approximately 34 open reading frames (ORFs). These Fusellovirus ORFs show little or no similarity to genes in the public databases. In contrast, 18 ORFs are common to all four isolates and may represent the minimal gene set defining this viral group. In general, ORFs on one half of the genome are colinear and highly conserved, while ORFs on the other half are not. One shared ORF among all four genomes is an integrase of the tyrosine recombinase family. All four viral genomes integrate into their host tRNA genes. The specific tRNA gene used for integration varies, and one genome integrates into multiple loci. Several unique ORFs are found in the genome of each isolate.

The preparation and subsequent imaging of a two-dimensional array of a genetically and chemically modified cowpea chlorotic mottle virus (CCMV) is described. The genetic mutation provides symmetrically dispersed exposed thiol groups on the outer surface of the virus capsid. These functional groups can be used to covalently bind the capsid to smooth Au substrate. AFM imaging suggests that the genetic mutation by itself does not promote array formation but, rather, aggregation through disulfide linkages. However, breaking the symmetry of the capsid using a solid-phase approach and chemically passivating the exposed thiol groups with iodoacetic acid results in a capsid with exposed thiols only on one side of the particle. These symmetry-broken capsids were able to form self-assembled monolayers (SAM) on a Au surface.

The protein cage of the 12-subunit ferritin-like protein from Listeria innocua has been utilized as a size and shape constrained reaction environment for the synthesis of two cobalt oxide minerals, Co(3)O(4) and Co(O)OH. Reaction of Co(II) with H(2)O(2) at pH 8.5 under either elevated temperature (65 degrees C) or ambient temperature (23 degrees C) resulted in the formation of cobalt oxide nanoparticles encapsulated within the protein cage. At elevated temperatures, Co(3)O(4) was formed while at lower temperature the oxyhydroxide Co(O)OH was found. Mineral particles, commensurate in size with the internal dimensions of the protein (5 nm), were imaged by transmission electron microscopy and shown to be surrounded by the intact protein cage. The minerals were investigated by electron diffraction and revealed a crystalline Co(3)O(4) phase and an amorphous Co(O)OH phase. Further investigation of these composite materials using size exclusion chromatography, gel electrophoresis, dynamic light scattering, and zeta potential measurements indicated that the mineral was encapsulated within the protein cage giving rise to properties of both the mineral and protein components.

The X-ray crystal structure of Cowpea chlorotic mottle bromovirus (CCMV) revealed a unique tubular structure formed by the interaction of the N-termini from six coat protein subunits at each three-fold axis of the assembled virion. This structure, termed the β-hexamer, consists of six short β-strands. The β-hexamer was postulated to play a critical role in the assembly and stability of the virion by stabilizing hexameric capsomers (Speir et al., 1995). Mutational analyses of the β-hexamer structure, utilizing both in vitro and in vivo assembly assays, demonstrate that this structure is not required for virion formation devoid of nucleic acids in vitro or for RNA-containing virions in vivo. However, the β-hexamer structure does contribute to virion stability in vitro and modulates disease expression in vivo. These results support a model for CCMV assembly through pentamer intermediates.

Cage architectures based on the cowpea chlorotic mottle virus (see Figure) have been employed to achieve a synthetic mimic of the iron storage protein ferritin. The electrostatic nature of the inner protein surface could be changed by up to 3240 units of charge, while still maintaining a stable cage structure. The spatial isolation within the protein cage prevents bulk aggregation of the mineral particles and results in a stable, mono-disperse colloid.

Homogeneous magnetic nanoparticles can be synthesised within the constrained volume architecture of Listeria innocua (see Figure). This ferritin-like protein cage provides a limited reaction environment for the synthesis of 5 nm diameter ferrimagnetic iron oxide maghemite nanoparticles. The results suggest a new general synthesis route for nanomaterials, utilizing the inherent host-guest properties of protein cage architectures.

Neutral pion photoproduction from a liquid deuterium target was measured in the energy region near 300 MeV at the LEGS facility of Brookhaven National Laboratory. The inclusive cross sections from deuterium are in agreement with measurments from Mainz, yet the exclusive cross sections and spin asymmetries for neutral pion production in coincidence with a detected nucleon are much smaller than expected from a quasi-free approximation. This may indicate that substantial final state interactions play a significant role, which will complicate the extraction of the desired amplitudes that would be measured if a free neutron target could be used.

Here we present generalized methods for chemically modifying the surface of a viral protein cage; this exploits the chemistry of native and engineered surface exposed functional groups for multivalent presentation of ligands.

Viruses of extreme thermophiles are of great interest because they serve as model systems for understanding the biochemistry and molecular biology required for life at high temperatures. In this work, we report the discovery, isolation, and preliminary characterization of viruses and virus-like particles from extreme thermal acidic environments (70–92°C, pH 1.0–4.5) found in Yellowstone National Park. Six unique particle morphologies were found in Sulfolobus enrichment cultures. Three of the particle morphologies are similar to viruses previously isolated from Sulfolobus species from Iceland and/or Japan. Sequence analysis of their viral genomes suggests that they are related to the Icelandic and Japanese isolates. In addition, three virus particle morphologies that had not been previously observed from thermal environments were found. These viruses appear to be completely novel in nature.

Cross sections are presented for the O-16(<(gamma)over right arrow>, pi(-) p) reaction at incident photon energies between 290 and 325 MeV. The data are presented for specific proton and pion angles as a function of proton energy, which are compared with calculations in a local distorted wave impulse approximation model. The results are in agree ment at most kinematics, although at some kinematics the data and calculations disagree by a factor of 2 or more. These data do not support the conclusion of a large modification to the mass of the Delta resonance in the nucleus.