The Cyanobacterial Repressor SmtB Is Predominantly a Dimer and Binds Two Zn2+ Ions per Subunit
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- 作者：Sambit R. Kar ; Amy C. Adams ; Jacob Lebowitz ; Kenneth B. Taylor ; and Leo M. Hall
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：December 9, 1997
- 年：1997
- 卷：36
- 期：49
- 页码：15343 - 15348
- 全文大小：109K
- 年卷期：v.36,no.49(December 9, 1997)
- ISSN：1520-4995
文摘
The Synechococcus PCC7942 metallothionein repressorgene smtB has been cloned into ahigh expression vector and the protein purified to near homogeneity(
98%). Analytical ultracentrifugationstudies demonstrate that the protein is predominantly dimeric in 0.1 MNaCl, pH 7.4, and 22 
C, exhibitinga monomer-dimer-tetramer equilibrium. The monomer-dimer(Ka(1,2)) and the dimer-tetramer(Ka(2,4))association constants are 3.24 × 105 and 9.90 ×102 M-1, respectively. Therepressor binds two Zn2+ions per subunit with an overall Kd of 3.49 ×10-6 M. In the presence ofZn2+, Ka(1,2) increases by2orders of magnitude to 1.25 × 107M-1 and the apparent weight-averagedsedimentation coefficient increasesfrom 2.00 to 2.22 S. The fact that the increase in sedimentationcoefficient is greater than that predictedby increased dimerization is interpreted as caused by compaction of thestructure in the presence of metalions. At pH 6.0, 0.1 M NaCl, and 22 C, the protein exhibitsonly a monomer-dimer equilibrium, withKa(1,2) = 1.52 × 107M-1 which is almost identical to that seenupon binding Zn2+ at pH 7.4. Thecompaction and conformational change in SmtB caused byZn2+ is consistent with a role for thisalteredquaternary state in derepression of smtA inSynechococcus challenged with heavy metal ions.
98%). Analytical ultracentrifugationstudies demonstrate that the protein is predominantly dimeric in 0.1 MNaCl, pH 7.4, and 22 C, exhibitinga monomer-dimer-tetramer equilibrium. The monomer-dimer(Ka(1,2)) and the dimer-tetramer(Ka(2,4))association constants are 3.24 × 105 and 9.90 ×102 M-1, respectively. Therepressor binds two Zn2+ions per subunit with an overall Kd of 3.49 ×10-6 M. In the presence ofZn2+, Ka(1,2) increases by2orders of magnitude to 1.25 × 107M-1 and the apparent weight-averagedsedimentation coefficient increasesfrom 2.00 to 2.22 S. The fact that the increase in sedimentationcoefficient is greater than that predictedby increased dimerization is interpreted as caused by compaction of thestructure in the presence of metalions. At pH 6.0, 0.1 M NaCl, and 22 C, the protein exhibitsonly a monomer-dimer equilibrium, withKa(1,2) = 1.52 × 107M-1 which is almost identical to that seenupon binding Zn2+ at pH 7.4. Thecompaction and conformational change in SmtB caused byZn2+ is consistent with a role for thisalteredquaternary state in derepression of smtA inSynechococcus challenged with heavy metal ions.