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1.  The C-terminal cysteine annulus participates in auto-chaperone function for Salmonella phage P22 tailspike folding and assembly 
Bacteriophage  2012;2(1):36-49.
Elongated trimeric adhesins are a distinct class of proteins employed by phages and viruses to recognize and bind to their host cells, and by bacteria to bind to their target cells and tissues. The tailspikes of E. coli phage K1F and Bacillus phage Ø29 exhibit auto-chaperone activity in their trimeric C-terminal domains. The P22 tailspike is structurally homologous to those adhesins. Though there are no disulfide bonds or reactive cysteines in the native P22 tailspikes, a set of C-terminal cysteines are very reactive in partially folded intermediates, implying an unusual local conformation in the domain. This is likely to be involved in the auto-chaperone function. We examined the unusual reactivity of C-terminal tailspike cysteines during folding and assembly as a potential reporter of auto-chaperone function. Reaction with IAA blocked productive refolding in vitro, but not off-pathway aggregation. Two-dimensional PAGE revealed that the predominant intermediate exhibiting reactive cysteine side chains was a partially folded monomer. Treatment with reducing reagent promoted native trimer formation from these species, consistent with transient disulfide bonds in the auto-chaperone domain. Limited enzymatic digestion and mass spectrometry of folding and assembly intermediates indicated that the C-terminal domain was compact in the protrimer species. These results indicate that the C-terminal domain of the P22 tailspike folds itself and associates prior to formation of the protrimer intermediate, and not after, as previously proposed. The C-terminal cysteines and triple β-helix domains apparently provide the staging for the correct auto-chaperone domain formation, needed for alignment of P22 tailspike native trimer.
PMCID: PMC3357383  PMID: 22666655
auto-chaperone; cysteines; folding intermediates; tailspike; transient disulfide bond
2.  Visualizing the structural changes of bacteriophage epsilon15 and its Salmonella host during infection 
Journal of molecular biology  2010;402(4):731-740.
The efficient mechanism by which double stranded DNA bacteriophages deliver their chromosome across the outer membrane, cell wall, and inner membrane of Gram-negative bacteria remains obscure. Advances in single particle electron cryo-microscopy have recently revealed details of the organization of the DNA injection apparatus within the mature virion for various bacteriophages, including epsilon15 (ε15) and P-SSP7. We have used electron cryo-tomography and 3D subvolume averaging to capture snapshots of ε15 infecting its host Salmonella anatum. These structures suggest the following stages of infection. In the first stage, the tailspikes of ε15 attach to the surface of the host cell. Next ε15's tail hub attaches to a putative cell receptor and establishes a tunnel through which the injection core proteins behind the portal exit the virion. A tube spanning the periplasmic space is formed for viral DNA passage, presumably from the rearrangement of core proteins or from cellular components. This tube would direct the DNA into the cytoplasm and protect it from periplasmic nucleases. Once the DNA has been injected into the cell, the tube and portal seals, and the empty bacteriophage remains at the cell surface.
PMCID: PMC3164490  PMID: 20709082
virus; infection; Salmonella; electron; cryo-tomography
3.  Intracellular Assembly of Cyanophage Syn5 Proceeds through a Scaffold-Containing Procapsid▿ †  
Journal of Virology  2010;85(5):2406-2415.
Syn5 is a marine cyanophage that is propagated on the marine photosynthetic cyanobacterial strain Synechococcus sp. WH8109 under laboratory conditions. Cryoelectron images of this double-stranded DNA (dsDNA) phage reveal an icosahedral capsid with short tail appendages and a single novel hornlike structure at the vertex opposite the tail. Despite the major impact of cyanophages on life in the oceans, there is limited information on cyanophage intracellular assembly processes within their photosynthetic hosts. The one-step growth curve of Syn5 demonstrated a short cycle with an eclipse period of ∼45 min, a latent phase of ∼60 min, and a burst size of 20 to 30 particles per cell at 28°C. SDS-PAGE and Western blot analysis of cell lysates at different times after infection showed the synthesis of major virion proteins and their increase as the infection progressed. The scaffolding protein of Syn5, absent from virions, was identified in the lysates and expressed from the cloned gene. It migrated anomalously on SDS-PAGE, similar to the phage T7 scaffolding protein. Particles lacking DNA but containing the coat and scaffolding proteins were purified from Syn5-infected cells using CsCl centrifugation followed by sucrose gradient centrifugation. Electron microscopic images of the purified particles showed shells lacking condensed DNA but filled with protein density, presumably scaffolding protein. These findings suggest that the cyanophages form infectious virions through the initial assembly of scaffolding-containing procapsids, similar to the assembly pathways for the enteric dsDNA bacteriophages. Since cyanobacteria predate the enteric bacteria, this procapsid-mediated assembly pathway may have originated with the cyanophages.
PMCID: PMC3067778  PMID: 21177804
4.  Thermolabile folding intermediates: inclusion body precursors and chaperonin substrates 
An unexpected aspect of the expression of cloned genes is the frequent failure of newly synthesized polypeptide chains to reach their native state, accumulating instead as insoluble inclusion bodies. Amyloid deposits represent a related state associated with a variety of human diseases. The critical folding intermediates at the juncture of productive folding and the off-pathway aggregation reaction have been identified for the phage P22 tailspike and coat proteins. Though the parallel β coil tailspike is thermostable, an early intracellular folding intermediate is thermolabile. As the temperature of intracellular folding is increased, this species partitions to inclusion bodies, a kinetic trap within the cell. The earliest intermediates along the in vitro aggregation pathway, sequential multimers of the thermolabile folding intermediates, have been directly identified by native gel electrophoresis. Temperature-sensitive folding (tsf) mutations identify sites in the β coil domain, which direct the junctional intermediate down the productive pathway. Global suppressors of tsf mutants inhibit the pathway to inclusion bodies, rescuing the mutant chains. These mutants identify sites important for avoiding aggregation. Coat folding intermediates also partition to inclusion bodies as temperature is increased. Coat tsf mutants are suppressed by overexpression of the GroE chaperonin, indicating that the thermolabile intermediate is a physiological substrate for GroE. We suggest that many proteins are likely to have thermolabile intermediates in their intracellular folding pathways, which will be precursors to inclusion body formation at elevated temperatures and therefore substrates for heat shock chaperonins.
PMCID: PMC2040114  PMID: 8566549
inclusion body; protein folding; chaperones; aggregation
5.  Protein Folding Failure Sets High-Temperature Limit on Growth of Phage P22 in Salmonella enterica Serovar Typhimurium 
The high-temperature limit for growth of microorganisms differs greatly depending on their species and habitat. The importance of an organism's ability to manage thermal stress is reflected in the ubiquitous distribution of the heat shock chaperones. Although many chaperones function to reduce protein folding defects, it has been difficult to identify the specific protein folding pathways that set the high-temperature limit of growth for a given microorganism. We have investigated this for a simple system, phage P22 infection of Salmonella enterica serovar Typhimurium. Production of infectious particles exhibited a broad maximum of 150 phage per cell when host cells were grown at between 30 and 39°C in minimal medium. Production of infectious phage declined sharply in the range of 40 to 41°C, and at 42°C, production had fallen to less than 1% of the maximum rate. The host cells maintained optimal division rates at these temperatures. The decrease in phage infectivity was steeper than the loss of physical particles, suggesting that noninfectious particles were formed at higher temperatures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a decrease in the tailspike adhesins assembled on phage particles purified from cultures incubated at higher temperatures. The infectivity of these particles was restored by in vitro incubation with soluble tailspike trimers. Examination of tailspike folding and assembly in lysates of phage-infected cells confirmed that the fraction of polypeptide chains able to reach the native state in vivo decreased with increasing temperature, indicating a thermal folding defect rather than a particle assembly defect. Thus, we believe that the folding pathway of the tailspike adhesin sets the high-temperature limit for P22 formation in Salmonella serovar Typhimurium.
PMCID: PMC492335  PMID: 15294822

Results 1-5 (5)