THE CYANOBACTERIAL REPRESSOR SMTB IS PREDOMINANTLY A DIMER AND BINDS 2 ZN2+ IONS PER SUBUNIT

The Synechococcus PCC7942 metallothionein repressor gene smtB has been cloned into a high expression vector and the protein purified to near homogeneity (greater than or equal to 98%). Analytical ultracentrifug ation studies demonstrate that the protein is predominantly dimeric in 0.1 M NaCl, pH 7.4, and 22 degrees C, exhibiting a monomer-dimer-tetr amer equilibrium. The monomer-dimer (K-a(1,K-2)) and the dimer-tetrame r (K-a(2,K-4)) association constants are 3.24 x 10(5) and 9.90 x 10(2) M-1, respectively, The repressor binds two Zn2+ ions per subunit with an overall K-d of 3.49 x 10(-6) M. In the presence of Zn2+, K-a(1,K-2 ) increases by 2 orders of magnitude to 1.25 x 10(7) M-1 and the appar ent weight-averaged sedimentation coefficient increases from 2.00 to 2 .22 S. The fact that the increase in sedimentation coefficient is grea ter than that predicted by increased dimerization is interpreted as ca used by compaction of the structure in the presence of metal ions, At pH 6.0, 0.1 M NaCl, and 22 degrees C, the protein exhibits only a mono mer-dimer equilibrium, with K-a(1,K-2) = 1.52 x 10(7) M-1 which is alm ost identical to that seen upon binding Zn2+ at pH 7.4, The compaction and conformational change in SmtB caused by Zn2+ is consistent with a role for this altered quaternary state in derepression of smtA in Syn echococcus challenged with heavy metal ions.