Global conformation of the Escherichia coli replication factor DnaC protein in absence and presence of nucleotide cofactors

Roberto Galletto, Rodrigo Maillard, Maria J. Jezewska, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Global conformational and oligomeric states of the Escherichia coli replicative factor DnaC protein in the absence and presence of magnesium and nucleotide cofactors, ATP and ADP, and their fluorescent analogues, MANT-ATP and MANT-ADP, have been examined using analytical sedimentation velocity and time-dependent fluorescence anisotropy techniques. In solution, the DnaC protein exists exclusively as a monomer over a large protein concentration range. The value of s20,w o = 2.45 ± 0.07 S indicates that the protein molecule has an elongated shape. When modeled as a prolate ellipsoid of revolution, the hydrated DnaC protein has an axial ratio of 4.0 ± 0.6 with long axis a = 112 Å and the short axis b = 28 Å. respectively. The presence of magnesium or nucleotide cofactors, ATP or ADP, does not affect the global conformation of the protein and its monomeric state. These data indicate that recently found cooperative interactions between the DnaC molecules, in the complex with the DnaB helicase, are induced by the binding to the helicase, i.e., they are not the intrinsic property of the DnaC protein. Fluorescence anisotropy decays of the DnaC-MANT-ATP and DnaC-MANT-ADP complexes indicate that the protein has a rigid global structure on the nanosecond time scale, little affected by the nucleotide cofactors. Nevertheless, the complex with ATP has a more flexible structure, while the complex with ADP is more rigid, with the protein molecule assuming a more elongated shape. Magnesium exerts control only on the complex with the ATP analogue. In the absence of magnesium, the ATP analogue is firmly held in the binding site. In the presence of Mg2+, this fixed location is released and the analogue is allowed to assume a flexible conformational state. The significance of the results for the functioning of the DnaC protein is discussed.

Original languageEnglish (US)
Pages (from-to)10988-11001
Number of pages14
JournalBiochemistry
Volume43
Issue number34
DOIs
StatePublished - Aug 31 2004

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Escherichia coli
Conformations
Nucleotides
Adenosine Triphosphate
Magnesium
Proteins
Adenosine Diphosphate
Fluorescence Polarization
DnaB Helicases
Phosmet
Molecules
Anisotropy
Protein Conformation
Fluorescence
Protein S
Flexible structures
Sedimentation
Binding Sites
Monomers

ASJC Scopus subject areas

  • Biochemistry

Cite this

Global conformation of the Escherichia coli replication factor DnaC protein in absence and presence of nucleotide cofactors. / Galletto, Roberto; Maillard, Rodrigo; Jezewska, Maria J.; Bujalowski, Wlodzimierz.

In: Biochemistry, Vol. 43, No. 34, 31.08.2004, p. 10988-11001.

Research output: Contribution to journalArticle

Galletto, Roberto ; Maillard, Rodrigo ; Jezewska, Maria J. ; Bujalowski, Wlodzimierz. / Global conformation of the Escherichia coli replication factor DnaC protein in absence and presence of nucleotide cofactors. In: Biochemistry. 2004 ; Vol. 43, No. 34. pp. 10988-11001.
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