Chemical and Physical Biology Program, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Biochemistry. 2010 Dec 28;49(51):10780-7. doi: 10.1021/bi101495x. Epub 2010 Dec 3.
We have characterized the self-association of FtsZ in its GDP-bound state (GDP-FtsZ) and the heteroassociation of FtsZ and a soluble recombinant ZipA (sZipA) lacking the N-terminal transmembrane domain by means of composition gradient-static light scattering (CG-SLS) and by measurement of sedimentation equilibrium. CG-SLS experiments at high ionic strengths and in the presence of 5 mM Mg(2+) show that, while FtsZ self-associates in a noncooperative fashion, sZipA acts as a monomer. CG-SLS data obtained from mixtures of FtsZ (A) and sZipA (B) in the presence of Mg(2+) are quantitatively described by an equilibrium model that takes into account significant scattering contributions from B, A(1), A(2), A(3), A(4), A(5), A(6), A(1)B, A(2)B, A(3)B, and A(4)B. However, in the absence of Mg(2+) (with EDTA), the data are best explained by an equilibrium model in which only B, A(1), A(2), A(3), A(1)B, and A(2)B contribute significantly to scattering. The best-fit molecular weights of monomeric A and B are in good agreement with values calculated from amino acid composition and with values obtained from sedimentation equilibrium. The latter technique also confirmed the interaction between sZipA and GDP-FtsZ. Moreover, the association model that best describes the CG-SLS data is in qualitative agreement with the sedimentation data. From these results, it follows that the binding of sZipA to GDP-FtsZ is of moderate affinity and does not significantly affect the interactions between FtsZ monomers. Under the working conditions used, only one sZipA binds to FtsZ oligomers with a length of six at most. The observed behavior would be compatible with FtsZ fibrils being anchored in vivo to the bacterial inner plasma membrane by substoichiometric binding of membrane-bound ZipA.
我们通过组成梯度静态光散射（CG-SLS）和沉降平衡测量，对 GDP 结合态 FtsZ（GDP-FtsZ）的自缔合以及 FtsZ 与缺乏 N 端跨膜结构域的可溶性重组 ZipA（sZipA）的异源缔合进行了表征。在高离子强度和存在 5 mM Mg 2+的情况下进行的 CG-SLS 实验表明，虽然 FtsZ 以非协同方式自缔合，但 sZipA 作为单体存在。在 Mg 2+存在下，从 FtsZ（A）和 sZipA（B）混合物中获得的 CG-SLS 数据通过一个平衡模型进行定量描述，该模型考虑了 B、A(1)、A(2)、A(3)、A(4)、A(5)、A(6)、A(1)B、A(2)B、A(3)B 和 A(4)B 的显著散射贡献。然而，在没有 Mg 2+（用 EDTA）的情况下，数据最好通过一个平衡模型来解释，该模型中只有 B、A(1)、A(2)、A(3)、A(1)B 和 A(2)B 对散射有显著贡献。单体 A 和 B 的最佳拟合分子量与氨基酸组成计算的值以及从沉降平衡获得的值非常吻合。后一种技术也证实了 sZipA 与 GDP-FtsZ 之间的相互作用。此外，描述 CG-SLS 数据的最佳关联模型与沉降数据在定性上是一致的。由此可以得出结论，sZipA 与 GDP-FtsZ 的结合具有中等亲和力，并且不会显著影响 FtsZ 单体之间的相互作用。在使用的工作条件下，只有一个 sZipA 与最多六个 FtsZ 寡聚物结合。观察到的行为与 FtsZ 原纤维在体内通过亚化学计量结合膜结合的 ZipA 锚定在细菌内膜的行为是一致的。
