ANALYSIS OF THE STOICHIOMETRY OF THE T4 GENE 45 PROTEIN BY ION-SPRAY MASS-SPECTROMETRY

The synthesis of bacteriophage T4 DNA is catalyzed by a complex of fiv e proteins that includes the T4 DNA polymerase protein, one single-str anded DNA binding protein, and three T4-coded polymerase accessory pro teins. The accessory proteins form two subassemblies, one consisting o f 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 bas is of equilibrium sedimentation, velocity sedimentation,and chemical c ross-linking studies, aqueous gp45 was thought to exist as a noncovale nt trimer at pH 7. However, independent studies employing a combinatio n of gel filtration and sucrose gradient sedimentation suggested that gp45 exists as a dimer in solution. We report an investigation into th e stoichiometry of gp45 association in solution using a combination of ion spray mass spectrometry (MS) and microsore size exclusion chromat ography. In 10 mM NH4OAc (pH 2.5), both homodimer and trimer were obse rved by ion spray MS. Further experiments on two representative gp45 d imer ions using tandem mass spectrometry confirmed their structures as noncovalent gasphase association complexes. Judging from size exclusi on 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.