Bentley A Fane

PMID: 10329166;Abstract:

An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage φX174. Assembly of the φX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of a 'closed' procapsid particle has been determined to 3.5 Å resolution. This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 Å resolution electron microscopic reconstruction of the 'open' procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly α-helical fold and displays remarkable conformational variability. We report here an improved and refined structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching.

Nectar-feeding animals have among the highest recorded metabolic rates. High aerobic performance is linked to oxidative damage in muscles. Antioxidants in nectar are scarce to nonexistent. We propose that nectarivores use nectar sugar to mitigate the oxidative damage caused by the muscular demands of flight. We found that sugar-fed moths had lower oxidative damage to their flight muscle membranes than unfed moths. Using respirometry coupled with δ13C analyses, we showed that moths generate antioxidant potential by shunting nectar glucose to the pentose phosphate pathway (PPP), resulting in a reduction in oxidative damage to the flight muscles. We suggest that nectar feeding, the use of PPP, and intense exercise are causally linked and have allowed the evolution of powerful fliers that feed on nectar.

PMID: 1648264;Abstract:

Amino acid substitutions at a site in the center of the bacteriophage protein P22 tailspike polypeptide chain suppress temperature-sensitive folding mutations at many sites throughout the chain. Characterization of the intracellular folding and chain assembly process reveals that the suppressors act in the folding pathway, inhibiting the aggregation of an early folding intermediate into the kinetically trapped inclusion body state. The suppressors alone increase the folding efficiency of the otherwise wild-type polypeptide chain without altering the stability or activity of the native state. These amino acid substitutions identify an unexpected aspect of the protein folding grammar-sequences within the chain that carry information inhibiting unproductive off-pathway conformations. Such mutations may serve to increase the recovery of protein products of cloned genes.

PMID: 18400861;PMCID: PMC2395140;Abstract:

In the φX174 procapsid crystal structure, 240 external scaffolding protein D subunits form 60 pairs of asymmetric dimers, D1D 2 and D3D4, in a non-quasi-equivalent structure. To achieve this arrangement, α-helix 3 assumes two different conformations: (i) kinked 30° at glycine residue 61 in subunits D 1 and D3 and (ii) straight in subunits D2 and D4. Substitutions for G61 may inhibit viral assembly by preventing the protein from achieving its fully kinked conformation while still allowing it to interact with other scaffolding and structural proteins. Mutations designed to inhibit conformational switching in α-helix 3 were introduced into a cloned gene, and expression was demonstrated to inhibit wild-type morphogenesis. The severity of inhibition appears to be related to the size of the substituted amino acid. For infections in which only the mutant protein is present, morphogenesis does not proceed past the first step that requires the wild-type external scaffolding protein. Thus, mutant subunits alone appear to have little or no morphogenetic function. In contrast, assembly in the presence of wild-type and mutant subunits is blocked prematurely, before D protein is required in a wild-type infection, or channeled into an off-pathway reaction. These data suggest that the wild-type protein transports the inhibitory protein to the pathway. Viruses resistant to the lethal dominant proteins were isolated, and mutations were mapped to the coat and internal scaffolding proteins. The affected amino acids cluster in the atomic structure and may act to exclude mutant subunits from occupying particular positions atop pentamers of the viral coat protein. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

PMID: 15046981;Abstract:

Packaging of viral genomes into their respective capsids requires partial neutralization of the highly negatively charged RNA or DNA. Many viruses, including the Microviridae bacteriophages φX174, G4, and α3, have solved this problem by coding for a highly positively charged nucleic acid-binding protein that is packaged along with the genome. The φX174 DNA-binding protein, J, is 13 amino acid residues longer than the α3 and G4 J proteins by virtue of an additional nucleic acid-binding domain at the amino terminus. Chimeric φX174 particles containing the smaller DNA-binding protein cannot be generated due to procapsid instability during DNA packaging. However, chimeric α3 and G4 phages, containing the φX174 DNA-binding protein in place of the endogenous J protein, assemble and are infectious, but are less dense than the respective wild-type species. In addition, host cell attachment and native gel migration assays indicate surface variations of these viruses that are controlled by the nature of the J protein. The structure of α3 packaged with φX174 J protein was determined to 3.5Å resolution and compared with the previously determined structures of φX174 and α3. The structures of the capsid and spike proteins in the chimeric particle remain unchanged within experimental error when compared to the wild-type α3 virion proteins. The amino-terminal region of the φX174 J protein, which is missing from wild-type α3 virions, is mostly disordered in the α3 chimera. The differences observed between solution properties of wild-type φX174, wild-type α3, and α3 chimera, including their ability to attach to host cells, correlates with the degree of order in the amino-terminal domain of the J protein. When ordered, this domain binds to the interior of the viral capsid and, thus, might control the flexibility of the capsid. In addition, the properties of the φX174 J protein in the chimera and the results of mutational analyses suggest that an evolutionary correlation may exist between the size of the J protein and the stoichiometry of the DNA pilot protein H, required in the initial stages of infection. Hence, the function of the J protein is to facilitate DNA packaging, as well as to mediate surface properties such as cell attachment and infection. © 2004 Elsevier Ltd. All rights reserved.