Enzymic and Molecular Properties of Base-Plate Parts of Bacteriophage P22

Iwashita, Shintaro; Kanegasaki, Shiro

doi:10.1111/j.1432-1033.1976.tb10392.x

Cited by 82 publications

(46 citation statements)

References 24 publications

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“…With incubation time, the exclusion peak broadens and is shifted toward smaller sizes before cleavage end products octa-and tetrasaccharides appear. These end products had also been observed earlier in preparations with the phage (30). They are a consequence of the special arrangement of catalytic residues that lie at the end of a high affinity binding pocket (Fig.…”

Section: P22 Dna Is Released Specifically Upon Contact With Lps Frommentioning

confidence: 75%

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Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Andres¹,

Hanke

Baxa³

et al. 2010

Journal of Biological Chemistry

138

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Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.

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Section: P22 Dna Is Released Specifically Upon Contact With Lps Frommentioning

confidence: 75%

“…Hydrolysis of Salmonella O-antigen by P22 TSP produces oligosaccharide fragments comprising at least two RU (30). We incubated polysaccharide with TSP and analyzed the kinetics of product formation using gel filtration (Fig.…”

Section: P22 Dna Is Released Specifically Upon Contact With Lps Frommentioning

confidence: 99%

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Andres¹,

Hanke

Baxa³

et al. 2010

Journal of Biological Chemistry

138

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“…The P22 tailspike protein, a homotrimer of 72 kDa (666 amino-acid residue) chains [14-161 is essential for phage adsorption and displays endorhamnosidase activity hydrolyzing the Salmonella 0-antigen polysaccharide [17]. The folding pathway of the tailspike in vivo [14], as well as during reconstitution in vitro [18], comprises consecutive subunit folding, subunit association, and a ratelimiting folding reaction at the trimer level.…”

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confidence: 99%

Folding and assembly of phage P22 tailspike endorhamnosidase lacking the N‐terminal, head‐binding domain

Danner

Fuchs

Miller

et al. 1993

European Journal of Biochemistry

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Tryptic digestion of a thermal unfolding intermediate of the phage P22 tailspike endorhamnosidase produces an N-terminally shortened protein fragment comprising amino-acid residues 108 -666 [Chen, B.-L. & King, J. (1991) Biochemistry 30, . In the present work, the 60-kDa C-terminal fragment was purified to homogeneity from the tryptic digest by gel-fitration chromatography. As is the case for the whole tailspike protein (72 kDa), the purified fragment was found to remain stably folded as a highly soluble, SDS-resistant, enzymatically active trimer. However, its unfolding in the presence of guanidinium chloride was accelerated at least 10-fold compared to the complete, native tailspike protein. Shortened tailspike trimers reconstituted spontaneously and with high yield after diluting a solution containing acid-urea-unfolded fragment polypeptides with neutral buffer. Upon recombinant expression of the 60-kDa polypeptide in Escherichia coli, it also assembled efficiently and formed SDS-resistant trimers. The refolding and assembly pathway of the Nterminally shortened tailspike paralleled that of the complete protein with slightly, but significantly, accelerated folding reactions, at both the subunit and the trimer levels. As found for the complete tailspike protein, yields of refolding and assembly of the 60-kDa fragments into SDS-resistant trimers decreased with increasing temperature. The refolding yield of fragments derived from a temperature-sensitive mutant (Gly244 + Arg) tailspike protein was affected in similar fashion as shown for the whole protein. We conclude that the N-terminal domain (residues 1-107) is dispensable for folding and assembly of the P22 tailspike endorhamnosidase both in vitro and in vivo.The native, functional structure of a folded protein is completely determined by the amino-acid sequence of its constitutent polypeptide chains [l -31. However, polypeptide sequence and three-dimensional structure are not connected by a simple, linear code, and attempts to predict the threedimensional structure of a protein from the amino-acid sequence have so far met with very limited success. There is general agreement that a successful structure prediction requires the understanding of protein folding pathways, and of the interactions stabilizing protein folding intermediates [4 -61. A number of observations suggest that protein folding intermediates are stabilized by a subset of the interactions present in the native protein structure [4, 7-91. In principle, however, parts of the amino-acid sequence may code for interactions only needed to guide the polypeptide through the correct folding pathway, but not used in the native, folded structure [lo-131.Observations on the effects of temperature-sensitive mutations in the gene coding for the tailspike protein of Salmonella typhimurium phage P22 have led to the proposal that the amino-acid sequence, indeed, contains information cru-

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“…The data show no obvious characteristic for this protein, but acidic residues are common, while for example glycine and branched chain aliphatic residues are less frequent. In some preparations of tail protein the total compositions showed a larger excess of glycine and leucine, thus resembling the amino acid composition given previously [4]. These preparations, however, were not quite reproducible and contained the tail protein in an apparently lower yield.…”

Section: Amino Acid Compositionmentioning

confidence: 73%

“…Some properties of the enzymatic tail protein of bacteriophage P22 were recently published [4] . In the present work chemical data on this protein are reported.…”

Section: North-holland Publishing Company -Amsterdammentioning

confidence: 99%