CHARACTERIZATION AND CRYSTALLISATION OF DivIVA MUTANT PROTEINS

Naďa Pavlendová, Katarína Muchová and Imrich Barák

Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava 45, Slovakia; phone: ++421 2 5930 7418, fax: ++421 2 5930 7416, e-mail: imrich.barak@savba.sk

Gram-positive bacterium Bacillus subtilis is the one of the most commonly studied model organism. Its life cycle represents a complex biological process. The mechanisms that ensure the correct positioning of the septum are complicated and still intensively studied. A machinery of proteins is involved.

DivIVA protein is a bifunctional protein [1]. During vegetative growth it acts as a controller of the mid-cell division site positioning. There is an inhibition complex of two proteins: MinC and MinD, which disable assembly of division machinery (first and foremost assembly of FtsZ ring), in positions where it occurs. A DivIVA protein arrests MinCD inhibition complex at the cell poles and thus releases the cell center for septum formation [1, 2, 3, 4]. During sporulation DivIVA is involved in chromosome segregation [1].

DivIVA is a 19,5 kDa tropomyosin-like a-helical coiled-coil protein. Cryonegative stain transmission electron microscopy revealed 3D structure of DivIVA consisting of particles with lateral expansions at both ends resembling a “doggy-bone”. These forms are about 145 kDa, which correspond to 6-8 mers. DivIVA oligomers can build higher structures like strings, wires and 2D nets [5].

It was found, that DivIVA amino acid residues 17, 18 and 19 are responsible for polar targeting of protein. So mutations at these sites may mimic conformational change that probably occurs and enables DivIVA its dual function [6]. We have cloned the divIVA localization mutant genes into expression vectors to prepare proteins for cryonegative stain transmission electron microscopy and for crystallization trials.

1. H.B. Thomaides, M. Freeman, M. El Karoui, J. Errington, Genes Dev., 15 (2001) 1662-1673.

2. D.H. Edwards and J. Errington, Mol. Microbiol. 24 (1997) 905-915

3. A.L. Marston, H.B. Thomaides, D.H. Edwards, M.E. Sharpe and J.Errington, Genes Dev., 12 (1998) 3419-3430

4. A.L. Marston and J. Errington, Mol. Microbiol,. 33 (1999) 84-96

5. H. Stahlberg, E. Kutejová, K. Muchová, M. Gregorini, A. Lustig, S.A. Müller, V. Olivieri, A. Engel, A.J Wilkinson and I. Barák, Mol. Microbiol., 52 (2004) 1281-1290

6. S.E. Perry and D.H. Edwards, Mol. Microbiol., 54(2004) 1237-1249

Work in author’s laboratory is supported by grant 2/1004/22 from the Slovak Academy of Sciences and Wellcome Trust Project and Collaborative Research Initiative Grants 066732/Z/01/Z.