The Escherichia coli primosomal dnat protein exists in solution as a monomer-trimer equilibrium system

Michal R. Szymanski, Maria J. Jezewska, Wlodzimierz Bujalowski

    Research output: Contribution to journalArticlepeer-review

    13 Scopus citations

    Abstract

    The oligomerization reaction of the Escherichia coli DnaT protein has been quantitatively examined using fluorescence anisotropy and analytical ultracentrifugation methods. In solution, DnaT exists as a monomer-trimer equilibrium system. At the estimated concentration in the E. coli cell, DnaT forms a mixture of the monomer and trimer states with a 3:1 molar ratio. In spite of the modest affinity, the trimerization is a highly cooperative process, without the detectable presence of the intervening dimer. The DnaT monomer consists of a large N-terminal core domain and a small C-terminal region. The removal of the C-terminal region dramatically affects the oligomerization process. The isolated N-terminal domain forms a dimer instead of the trimer. These results indicate that the DnaT monomer possesses two structurally different, interacting sites. One site is located on the N-terminal domain, and two monomers, in the trimer, are associated through their binding sites located on that domain. The C-terminal region forms the other interacting site. The third monomer is engaged through the C-terminal regions. Surprisingly, the high affinity of the N-terminal domain dimer indicates that the DnaT monomer undergoes a conformational transition upon oligomerization, involving the C-terminal region. These data and the high specificity of the trimerization reaction, i.e., lack of any oligomers higher than a trimer, indicate that each monomer in the trimer is in contact with the two remaining monomers. A model of the global structure of the DnaT trimer based on the thermodynamic and hydrodynamic data is discussed.

    Original languageEnglish (US)
    Pages (from-to)1845-1857
    Number of pages13
    JournalBiochemistry
    Volume52
    Issue number11
    DOIs
    StatePublished - Mar 19 2013

    ASJC Scopus subject areas

    • Biochemistry

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