Montecinos-Franjola Felipe, James Nicholas G, Concha-Marambio Luis, Brunet Juan E, Lagos Rosalba, Monasterio Octavio, Jameson David M
Laboratorio de Biología Estructural y Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 7800024, Chile.
Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu 96813, HI, USA.
Biochim Biophys Acta. 2014 Jul;1844(7):1193-200. doi: 10.1016/j.bbapap.2014.03.012. Epub 2014 Apr 2.
Cell division protein FtsZ cooperatively self-assembles into straight filaments when bound to GTP. A set of conformational changes that are linked to FtsZ GTPase activity are involved in the transition from straight to curved filaments that eventually disassemble. In this work, we characterized the fluorescence of single Trp mutants as a reporter of the predicted conformational changes between the GDP- and GTP-states of Escherichia coli FtsZ. Steady-state fluorescence characterization showed the Trp senses different environments and displays low solvent accessibility. Time-resolved fluorescence data indicated that the main conformational changes in FtsZ occur at the interaction surface between the N and C domains, but also minor rearrangements were detected in the bulk of the N domain. Surprisingly, despite its location near the bottom protofilament interface at the C domain, the Trp 275 fluorescence lifetime did not report changes between the GDP and GTP states. The equilibrium unfolding of FtsZ features an intermediate that is stabilized by the nucleotide bound in the N-domain as well as by quaternary protein-protein interactions. In this context, we characterized the unfolding of the Trp mutants using time-resolved fluorescence and phasor plot analysis. A novel picture of the structural transition from the native state in the absence of denaturant, to the solvent-exposed unfolded state is presented. Taken together our results show that conformational changes between the GDP and GTP states of FtsZ, such as those observed in FtsZ unfolding, are restricted to the interaction surface between the N and C domains.
细胞分裂蛋白FtsZ与GTP结合时会协同自组装成直丝。一系列与FtsZ GTPase活性相关的构象变化参与了从直丝到最终解聚的曲丝的转变。在这项工作中，我们将单个色氨酸突变体的荧光作为大肠杆菌FtsZ的GDP态和GTP态之间预测构象变化的报告指标进行了表征。稳态荧光表征表明，色氨酸感知不同环境并表现出低溶剂可及性。时间分辨荧光数据表明，FtsZ的主要构象变化发生在N结构域和C结构域之间的相互作用表面，但在N结构域主体中也检测到了微小的重排。令人惊讶的是，尽管色氨酸275位于C结构域底部原丝界面附近，但其荧光寿命并未显示GDP态和GTP态之间的变化。FtsZ的平衡解折叠具有一个中间体，该中间体由结合在N结构域中的核苷酸以及四级蛋白质-蛋白质相互作用稳定。在此背景下，我们使用时间分辨荧光和相量图分析对色氨酸突变体的解折叠进行了表征。呈现了从无变性剂时的天然状态到溶剂暴露的解折叠状态的结构转变的新图景。综合我们的结果表明，FtsZ的GDP态和GTP态之间的构象变化，如在FtsZ解折叠中观察到的那些变化，仅限于N结构域和C结构域之间的相互作用表面。
