INHIBITION OF ASSEMBLY OF BACTERIAL-CELL DIVISION PROTEIN FTSZ BY THEHYDROPHOBIC DYE 5,5'-BIS-(8-ANILINO-1-NAPHTHALENESULFONATE)

To gain further insight into the structural relatedness of tubulin and FtsZ, the tubulin-like prokaryotic cell division protein, we tested t he effect of tubulin assembly inhibitors on FtsZ assembly. Common tubu lin inhibitors, such as colchicine, colcemid, benomyl, and vinblastine , had no effect on Ca2+-promoted GTP-dependent assembly of FtsZ into p olymers. However, the hydrophobic probe 5,5'-bis-(8-anilino-1-naphthal enesulfonate) (bis-ANS) inhibited FtsZ assembly. The potential mechani sms for inhibition are discussed. Titrations of FtsZ with bis-ANS indi cated that FtsZ has one high affinity binding site an multiple low aff inity binding sites. ANS (8-anilino-1-naphthalenesulfonate), a hydroph obic probe similar to bis-ANS, had no inhibitory effect on FtsZ assemb ly. Because tubulin assembly has also been shown to be inhibited by bi s-ANS but not by ANS, it supports the idea that FtsZ and tubulin share similar conformational properties. Ca2+, which promotes GTP-dependent FtsZ assembly, stimulated binding of bis-ANS or ANS to FtsZ, suggesti ng that Ca2+ binding induces changes in the hydrophobic conformation o f the protein. Interestingly, depletion of bound Ca2+ with EGTA furthe r enhanced bis-ANS fluorescence. These findings suggest that both bind ing and dissociation of Ca2+ are capable of inducing FtsZ conformation al changes, and these changes could promote the GTP dependent assembly of FtsZ.