Structural transitions during bacteriophage HK97 head assembly

Structural transitions during bacteriophage HK97 head assembly Bacteriophage HK97 builds its head shell from a 42 kDa major head protein, but neither this 42 kDa protein nor its processed, 31 kDa form is found in the mature head. Instead, each of the major head-protein subunits is covalently cross-linked into oligomers of five, six or more by a protein cross-linking reaction that occurs both in vivo and in vitro . Mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed Prohead I and Prohead II. Prohead I is assembled from about 415 copies of the 42 kDa (384 amino acids) protein subunit and accumulates in infections by mutant am U4. Following assembly, the N-terminal 102 amino acids of each subunit are removed, leaving a prohead shell constructed of 31 kDa subunits, called Prohead II, which accumulates in infections by mutant am C2. During DNA packaging, when the prohead shell expands, all of the head protein subunits become covalently cross-linked to other subunits. Purified Prohead II (or, less completely, Prohead I) becomes cross-linked in vitro in response to any of a number of conditions that induce shell expansion, including conditions commonly used for protein analysis. In vitro cross-linking occurs efficiently in the absence of added cofactors of enzymes, and we propose that cross-linking is catalyzed by shell subunits themselves. Shell expansion is easily monitored by observing a decrease in electrophoretic mobility of Prohead II in agarose gels. Using the mobility shift in agarose gel to monitor expansion and SDS/gel electrophoresis to monitor cross-linking in vitro , we find that expansion precedes and is required for cross-linking, and we propose that expansion triggers the cross-linking reaction. Comparison of peptides isolated from Prohead II and in vitro cross-linked Prohead II shows a single altered major cross-link peptide in which a lysine, originating from lysine169 of the protein sequence, is linked to asparagine356, presumably derived from the neighboring subunit. Examination of the cross-link-containing peptide by mass spectrometry shows that the cross-link bond is an amide between the side-chains of the lysine and the asparagine residues. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Biology Elsevier

Structural transitions during bacteriophage HK97 head assembly

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Publisher
Elsevier
Copyright
Copyright © 1995 Academic Press Limited
ISSN
0022-2836
D.O.I.
10.1016/S0022-2836(05)80143-3
Publisher site
See Article on Publisher Site

Abstract

Bacteriophage HK97 builds its head shell from a 42 kDa major head protein, but neither this 42 kDa protein nor its processed, 31 kDa form is found in the mature head. Instead, each of the major head-protein subunits is covalently cross-linked into oligomers of five, six or more by a protein cross-linking reaction that occurs both in vivo and in vitro . Mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed Prohead I and Prohead II. Prohead I is assembled from about 415 copies of the 42 kDa (384 amino acids) protein subunit and accumulates in infections by mutant am U4. Following assembly, the N-terminal 102 amino acids of each subunit are removed, leaving a prohead shell constructed of 31 kDa subunits, called Prohead II, which accumulates in infections by mutant am C2. During DNA packaging, when the prohead shell expands, all of the head protein subunits become covalently cross-linked to other subunits. Purified Prohead II (or, less completely, Prohead I) becomes cross-linked in vitro in response to any of a number of conditions that induce shell expansion, including conditions commonly used for protein analysis. In vitro cross-linking occurs efficiently in the absence of added cofactors of enzymes, and we propose that cross-linking is catalyzed by shell subunits themselves. Shell expansion is easily monitored by observing a decrease in electrophoretic mobility of Prohead II in agarose gels. Using the mobility shift in agarose gel to monitor expansion and SDS/gel electrophoresis to monitor cross-linking in vitro , we find that expansion precedes and is required for cross-linking, and we propose that expansion triggers the cross-linking reaction. Comparison of peptides isolated from Prohead II and in vitro cross-linked Prohead II shows a single altered major cross-link peptide in which a lysine, originating from lysine169 of the protein sequence, is linked to asparagine356, presumably derived from the neighboring subunit. Examination of the cross-link-containing peptide by mass spectrometry shows that the cross-link bond is an amide between the side-chains of the lysine and the asparagine residues.

Journal

Journal of Molecular BiologyElsevier

Published: Apr 7, 1995

References

  • The DNA translocating vertex of dsDNA bacteriophage
    Bazinet, C.; King, J.
  • Genetic basis of bacteriophage HK97 prohead assembly
    Duda, R.L.; Martincic, K.; Hendrix, R.W.
  • Bacteriophage HK97 head assembly
    Duda, R.L.; Martincic, K.; Xie, Z.; Hendrix, R.W.
  • Transglutaminases
    Folk, J.E.
  • DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics
    Hatfull, G.F.; Sarkis, G.J.

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