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J. Biol. Chem., Vol. 281, Issue 18, 12526-12534, May 5, 2006

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Membrane-catalyzed Nucleotide Exchange on DnaA

EFFECT OF SURFACE MOLECULAR CROWDING^*

Alexander Aranovich, Garik Y. Gdalevsky, Rivka Cohen-Luria, Itzhak Fishov¹, and Abraham H. Parola²

From the Departments of Life Sciences and Chemistry, Ben-Gurion University of the Negev, P. O. B. 653, Beer-Sheva 84105, Israel

DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a central role in the timing of the primary initiations within the Escherichia coli cell cycle. A controlled, reversible conversion between the active ATP-DnaA and the inactive ADP forms modulates this activity. In a DNA-dependent manner, bound ATP is hydrolyzed to ADP. Acidic phospholipids with unsaturated fatty acids are capable of reactivating ADP-DnaA by promoting the release of the tightly bound ADP. The nucleotide dissociation kinetics, measured in the present study with the fluorescent derivative 3'-O-(N-methylantraniloyl)-5'-adenosine triphosphate, was dependent on the density of DnaA on the membrane in a cooperative manner: it increased 5-fold with decreased protein density. At all surface densities the nucleotide was completely released, presumably due to protein exchange on the membrane. Distinct temperature dependences and the effect of the crowding agent Ficoll suggest that two functional states of DnaA exist at high and low membrane occupancy, ascribed to local macromolecular crowding on the membrane surface. These novel phenomena are thought to play a major role in the mechanism regulating the initiation of chromosomal replication in bacteria.

Received for publication, September 19, 2005 , and in revised form, March 3, 2006.

^* This work was supported by the Israel Science Foundation (Grant 512/02 to A. H. P. and I. F.) and by the James Frank Foundation (to A. H. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed. Tel.: 972-8-646-1368; Fax: 972-8-646-1710; E-mail: fishov{at}bgu.ac.il. ² To whom correspondence may be addressed. Tel.: 972-8-647-2454; Fax: 972-8-647-2954; E-mail: aparola{at}bgu.ac.il.

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