Surface-accessible residues in the monomeric and assembled forms of a bacterial surface layer protein
Surface-accessible residues in the monomeric and assembled forms of a bacterial surface layer protein
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DOI：
10.1074/jbc.m003838200
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发表时间：
2000-12-01
影响因子：
4.8
通讯作者：
Bayley, H
中科院分区：
文献类型：
--
作者：
Howorka, S;Sára, M;Bayley, H
嗜热、革兰氏阳性生物脂肪嗜热芽孢杆菌PV72/p2的S-层蛋白SBSB形成一个结晶的、多孔的阵列，构成细胞被膜的最外层成分。SBSb的分子质量为98 kDa，对应的S层具有斜晶格对称性。为了研究SBSB的分子结构和组装，我们用半胱氨酸取代了75个残基(主要是丝氨酸、苏氨酸和丙氨酸)，这些残基分布在初级序列中，而野生型蛋白中没有半胱氨酸。经电子显微镜检测，75个突变体中有72个形成了与野生型相同的规则结构的自组装产物，从而证明了半胱氨酸取代了大部分选定的氨基酸并没有显著改变蛋白质的结构。然而，这三个有缺陷的突变体，其自组装能力大大降低，被成功地整合到了S的野生型蛋白质层中。通过5-kDa亲水性半胱氨酸-反应性聚乙二醇偶联物对单体SBSB突变体和组装成S层的SBSB突变体进行表面可及性筛选。在单体形式的SBSB中，34个被检查的残留物不能从表面进入，23个被归类为非常可进入的，18个被归类为中等表面可达的。相比之下，在组装的Sayers中，57个突变残基不可访问，6个非常可访问，12个中等可访问。结合其他结构信息，结果提出了SBSB的一个模型，其中功能结构域沿着多肽链的长度分离。
The S-layer protein SbsB of the thermophilic, Grampositive organism Bacillus stearothermophilus PV72/p2 forms a crystalline, porous array constituting the outermost component of the cell envelope. SbsB has a molecular mass of 98 kDa, and the corresponding S-layer exhibits an oblique lattice symmetry. To investigate the molecular structure and assembly of SbsB, we replaced 75 residues (mainly serine, threonine, and alanine), located throughout the primary sequence, with cysteine, which is not found in the wild-type protein. As determined by electron microscopy, 72 out of 75 mutants formed regularly-structured self-assembly products identical to wild-type, thereby proving that the replacement of most of the selected amino acids by cysteine does not dramatically alter the structure of the protein. The three defective mutants, which showed a greatly reduced ability to self-assemble, were, however, successfully incorporated into S-layers of wild-type protein. Monomeric SbsB mutants and SbsB mutants assembled into S-layers were subjected to a surface accessibility screen by targeted chemical modification with a 5-kDa hydrophilic cysteine-reactive polyethylene glycol conjugate. In the monomeric form of SbsB, 34 of the examined residues were not surface accessible, while 23 were classified as very accessible, and 18 were of intermediate surface accessibility. By contrast, in the assembled Slayers, 57 of the mutated residues were not accessible, six were very accessible, and 12 of intermediate accessibility. Together with other structural information, the results suggest a model for SbsB in which functional domains are segregated along the length of the polypeptide chain.