Instituto de Química-Física Rocasolano, Consejo Superior de Investigaciones Científicas, E-28006 Madrid, Spain.
Anal Biochem. 2011 Nov 1;418(1):89-96. doi: 10.1016/j.ab.2011.07.001. Epub 2011 Jul 13.
We present here a fluorescence anisotropy method for the quantification of the polymerization of FtsZ, an essential protein for cytokinesis in prokaryotes whose GTP-dependent assembly initiates the formation of the divisome complex. Using Alexa 488 labeled wild-type FtsZ as a tracer, the assay allows determination of the critical concentration of FtsZ polymerization from the dependence of the measured steady-state fluorescence anisotropy on the concentration of FtsZ. The incorporation of the labeled protein into FtsZ polymers and the lack of spectral changes on assembly were independently confirmed by time-resolved fluorescence and fluorescence correlation spectroscopy. Critical concentration values determined by this new assay are compatible with those reported previously under the same conditions by other well-established methods. As a proof of principle, data on the sensitivity of the assay to changes in FtsZ assembly in response to Mg(2+) concentration or to the presence of high concentrations of Ficoll 70 as crowding agent are shown. The proposed method is sensitive, low sample consuming, rapid, and reliable, and it can be extended to other cooperatively polymerizing systems. In addition, it can help to discover new antimicrobials that may interfere with FtsZ polymerization because it can be easily adapted to systematic screening assays.
我们在这里提出了一种荧光各向异性方法，用于定量聚合 FtsZ，这是原核生物细胞分裂所必需的蛋白质，其 GTP 依赖性组装起始于分裂体复合物的形成。使用 Alexa 488 标记的野生型 FtsZ 作为示踪剂，该测定法可以根据稳态荧光各向异性与 FtsZ 浓度的依赖性来确定 FtsZ 聚合的关键浓度。通过时间分辨荧光和荧光相关光谱学，独立证实了标记蛋白掺入 FtsZ 聚合物中以及组装过程中没有光谱变化。通过该新测定法确定的临界浓度值与其他先前在相同条件下通过其他成熟方法报道的值相兼容。作为原理验证，显示了该测定法对 FtsZ 组装响应 Mg（2+）浓度或高浓度 Ficoll 70 作为拥挤剂变化的灵敏度的数据。所提出的方法灵敏、样品消耗少、快速且可靠，并且可以扩展到其他协同聚合系统。此外，它可以帮助发现可能干扰 FtsZ 聚合的新抗菌药物，因为它可以很容易地适应系统筛选测定法。
