Background

Structural studies of a Salmonella Typhimurium flagellin protein indicated that four polar or charged C-terminal amino acid residues line the inner channel of the flagellum. The hydrophilic character of these putative channel-lining residues was predicted to be essential to facilitate the transport of unfolded flagellin monomers during flagellar assembly. The structure-function relationship of these putative channel-lining residues was investigated by site-directed mutagenesis to examine effects of side chain polarity and size on flagella assembly and function.

Methods

Channel-lining residue variants were generated using site-directed mutagenesis to substitute alanine and other residues to examine the effects of altered side-chain polarity on export and assembly. The export, in vivo motility function, and flagellar structure of variants was characterized by agar motility, video microscopy, immunofluorescence, and SDS-PAGE.

Results

Alanine substitution yielded decreased motility and flagellar assembly for three of the four residues. However, alanine substitution of residue Arg 494 did not alter export, although substitution with negatively charged glutamate decreased motility and flagellar filament length. Furthermore, many of the C-terminal mutations affected flagellar filament morphology and stability, often resulting in more tightly coiled and/or more brittle flagella than the wild type.

Conclusions

The four channel-lining C-terminal residues may facilitate monomer protein transport but also have structural roles in determining the stability and morphology of the flagellum.

General significance

These results provide further insight into the complex process of bacterial flagellin export and flagellar assembly and provide evidence of previously unknown structural functions for the four putative channel-lining residues.

中文翻译：

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预计在沙门氏菌鞭毛蛋白出口通道中排列的C末端残基的结构功能研究

背景

鼠伤寒沙门氏菌鞭毛蛋白的结构研究表明，鞭毛内部通道排列着四个极性或带电的C末端氨基酸残基。这些推定的通道衬里残基的亲水特性据预测对于鞭毛组装过程中促进未折叠鞭毛蛋白单体的运输至关重要。通过定点诱变研究这些推定的通道衬里残基的结构-功能关系，以检查侧链极性和大小对鞭毛装配和功能的影响。

方法

使用定点诱变替代丙氨酸和其他残基来研究通道侧残基变体，以检查改变的侧链极性对输出和组装的影响。变体的输出，体内运动功能和鞭毛结构的特征是琼脂运动，视频显微镜，免疫荧光和SDS-PAGE。

结果

丙氨酸取代导致四个残基中的三个残基的运动性和鞭毛装配降低。但是，残基Arg 494的丙氨酸取代不会改变输出，尽管用带负电荷的谷氨酸取代会降低运动性和鞭毛长度。此外，许多C末端突变影响鞭毛丝形态和稳定性，通常导致鞭毛卷曲和/或脆性较野生型更脆弱。

结论

四个通道衬里的C末端残基可以促进单体蛋白的运输，但在确定鞭毛的稳定性和形态方面也具有结构性作用。

一般意义

这些结果为细菌鞭毛蛋白输出和鞭毛组装的复杂过程提供了进一步的见识，并为四个推定的通道衬里残基提供了以前未知的结构功能的证据。