Efimov V P, Nepluev I V, Sobolev B N, Zurabishvili T G, Schulthess T, Lustig A, Engel J, Haener M, Aebi U
Ivanovsky Institute of Virology, Moscow, Russia.
J Mol Biol. 1994 Sep 30;242(4):470-86. doi: 10.1006/jmbi.1994.1595.
The bacteriophage T4 late gene wac (whisker's antigen control) encodes a fibrous protein which forms a collar/whiskers complex. Whiskers function as a helper protein for the long tail fibres assembly and plays a role in regulating retraction of the long tail fibres in response to environmental conditions. In this work we show that expression of the cloned wac gene in Escherichia coli yields a protein oligomer of 53 nm length which we call fibritin, and which is able to complement gpwac T4 particles in vitro. CD spectroscopy of fibritin indicates a 90% alpha-helical content, and scanning calorimetry shows that the protein has several distinct domains. The analysis of the 486 amino acid sequence of fibritin reveals three structural components: a 408 amino acid region that contains 12 putative coiled-coil segments with a canonical heptad (a-b-c-d-e-f-g)n substructure where the "a" and "d" positions are preferentially occupied by apolar residues, and the N and C-terminal domains (47 and 29 amino acid residues, respectively) have no heptad substructure. The distribution of hydrophobic residues within heptads is more similar to a triple than to a double coiled-coil. The alpha-helical segments are separated by short "linker" regions, variable in length, that have a high proportion of glycine and proline residues. Each coiled-coil segment has, on the borders with linker regions, residues that are common to the N and C-terminal caps of the alpha-helices. Full-length and amino-terminally truncated fibritins can be reassembled in vitro after temperature-induced denaturation. Co-assembly of full-length fibritin and the N-terminal deletion mutant, as well as analytical centrifugation, indicates that the protein is a parallel triple-standard alpha-helical coiled-coil. Deletions of various N-terminal portions of fibritin did not block trimerisation but the mutant trimers are unable to bind to T4 particles. The last 18 C-terminal residues of fibritin are required for correct trimerisation of gpwac monomers in vivo. We propose that fibritin might serve as a convenient model for the investigation of folding and assembly mechanisms of alpha-fibrous proteins.
噬菌体T4晚期基因wac（须状抗原控制基因）编码一种纤维状蛋白，该蛋白形成一个衣领/须状复合体。须状蛋白作为长尾纤维组装的辅助蛋白，在响应环境条件调节长尾纤维的缩回过程中发挥作用。在这项研究中，我们发现克隆的wac基因在大肠杆菌中表达产生一种长度为53纳米的蛋白质寡聚体，我们将其称为纤维蛋白，并且该蛋白能够在体外互补gpwac T4颗粒。纤维蛋白的圆二色光谱表明其α-螺旋含量为90%，扫描量热法显示该蛋白有几个不同的结构域。对纤维蛋白486个氨基酸序列的分析揭示了三个结构成分：一个408个氨基酸的区域，包含12个假定的卷曲螺旋片段，具有典型的七肽（a-b-c-d-e-f-g）n亚结构，其中“a”和“d”位置优先被非极性残基占据，N端和C端结构域（分别为47和29个氨基酸残基）没有七肽亚结构。七肽内疏水残基的分布更类似于三聚体卷曲螺旋而不是二聚体卷曲螺旋。α-螺旋片段由短的“连接”区域隔开，这些区域长度可变，含有高比例的甘氨酸和脯氨酸残基。每个卷曲螺旋片段在与连接区域的边界处，具有α-螺旋N端和C端帽共同的残基。温度诱导变性后，全长和氨基端截短的纤维蛋白可以在体外重新组装。全长纤维蛋白和N端缺失突变体的共组装以及分析离心表明该蛋白是一种平行的三链α-螺旋卷曲螺旋。纤维蛋白不同N端部分的缺失并不阻碍三聚化，但突变三聚体无法与T4颗粒结合。纤维蛋白的最后18个C端残基是体内gpwac单体正确三聚化所必需的。我们认为纤维蛋白可能是研究α-纤维状蛋白折叠和组装机制的一个方便模型。
