Vonderviszt F, Závodszky P, Ishimura M, Uedaira H, Namba K
International Institute for Advanced Research Matsushita Electric Industrial Co., Ltd., Seika, Japan.
J Mol Biol. 1995 Aug 25;251(4):520-32. doi: 10.1006/jmbi.1995.0453.
The terminal regions of monomeric hook protein from Salmonella typhimurium are known to be highly mobile and exposed to the solvent. Although hook protein exhibits an unusual far-UV circular dichroism spectrum, resembling that of random coil structures, our calorimetric experiments clearly demonstrate that the molecule has a compact ordered core. The compact part probably consists of three domains as suggested by deconvolution analysis of the calorimetric melting profiles. Secondary structure prediction, together with the analysis of far-UV circular dichroism spectra, has shown that the domains of monomeric hook protein contain beta-sheeted structures without significant alpha-helical content. The polymerization of hook protein is accompanied by the stabilization of its disordered terminal regions into a predominantly alpha-helical domain. Evaluation of circular dichroism data suggests that about 45 terminal residues are involved in helical segments. Coiled-coil prediction indicates that whereas the whole carboxy-terminal helical region of hook protein has a strong bundle-forming potential, there is only a single short amino-terminal segment exhibiting weak coiled-coil forming tendencies. The formation of alpha-helical bundles is commonly believed to be a key event during the polymerization of the axial structure of bacterial flagella. To clarify the role of helical bundle formation in hook assembly, proteolytic fragments of hook protein with truncations of various lengths in their carboxy-terminal disordered regions were generated, and their polymerization behavior was investigated. We found that even fragments completely lacking the main helix-forming carboxy-terminal regions can polymerize into filaments in vitro under appropriately high salt concentrations. Our results suggest that, although helical bundle formation may occur during self-assembly, governing precise subunit packing and playing an important role in the stabilization of hook filaments, it is not the principal interaction mainly responsible for the development of their filamentous structure.
已知鼠伤寒沙门氏菌单体钩蛋白的末端区域具有高度的流动性且暴露于溶剂中。尽管钩蛋白呈现出异常的远紫外圆二色光谱，类似于无规卷曲结构，但我们的量热实验清楚地表明该分子具有紧密有序的核心。如量热熔解曲线的去卷积分析所示，紧密部分可能由三个结构域组成。二级结构预测以及远紫外圆二色光谱分析表明，单体钩蛋白的结构域包含β折叠结构，α螺旋含量不显著。钩蛋白的聚合伴随着其无序末端区域稳定形成主要为α螺旋的结构域。对圆二色性数据的评估表明，约45个末端残基参与螺旋片段的形成。卷曲螺旋预测表明，虽然钩蛋白的整个羧基末端螺旋区域具有很强的束状形成潜力，但只有一个短的氨基末端片段表现出较弱的卷曲螺旋形成倾向。通常认为α螺旋束的形成是细菌鞭毛轴向结构聚合过程中的关键事件。为了阐明螺旋束形成在钩组装中的作用，我们生成了在其羧基末端无序区域具有不同长度截短的钩蛋白蛋白水解片段，并研究了它们的聚合行为。我们发现，即使是完全缺乏主要螺旋形成羧基末端区域的片段，在适当高盐浓度下也能在体外聚合成细丝。我们的结果表明，虽然螺旋束形成可能在自组装过程中发生，控制精确的亚基堆积并在钩细丝的稳定中起重要作用，但它不是主要负责其丝状结构形成的主要相互作用。
