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 journalArticle

    12 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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