Analysis of the Stoichiometry of the T4 Gene 45 Protein by Ion Spray Mass Spectrometry

Bruce Ganem, Yu Tsyr Li, Yin Liang Hsieh, Jack D. Henion, Barbara F. Kaboord, Michelle W. Frey, Stephen J. Benkovic

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

The synthesis of bacteriophage T4 DNA is catalyzed by a complex of five proteins that includes the T4 DNA polymerase protein, one single-stranded DNA binding protein, and three T4-coded polymerase accessory proteins. The accessory proteins form two subassemblies, one consisting of a tightly-bound complex of gene 44 and 62 proteins and the other an oligomer of the gene 45 protein (product of gene 45, gp45). On the basis of equilibrium sedimentation, velocity sedimentation, and chemical cross-linking studies, aqueous gp45 was thought to exist as a noncovalent trimer at pH 7. However, independent studies employing a combination of gel filtration and sucrose gradient sedimentation suggested that gp45 exists as a dimer in solution. We report an investigation into the stoichiometry of gp45 association in solution using a combination of ion spray mass spectrometry (MS) and microbore size exclusion chromatography. In 10 mM NH4OAc (pH 2.5), both homodimer and trimer were observed by ion spray MS. Further experiments on two representative gp45 dimer ions using tandem mass spectrometry confirmed their structures as noncovalent gasphase association complexes. Judging from size exclusion chromatography, a gp45 homotrimer was the predominant species in 10 mM sodium phosphate at pH 6.8, although a corroborating mass spectrum could not be obtained at that pH.

Original languageEnglish (US)
Pages (from-to)1352-1358
Number of pages7
JournalJournal of the American Chemical Society
Volume116
Issue number4
DOIs
StatePublished - Feb 1 1994

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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