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首页> 外文期刊>Journal of bacteriology >Surface Location of Individual Residues of SlpA Provides Insight into the Lactobacillus brevis S-Layer
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Surface Location of Individual Residues of SlpA Provides Insight into the Lactobacillus brevis S-Layer

机译：SlpA单个残基的表面位置提供了对短乳杆菌S层的洞察力

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摘要

Bacterial surface layer (S-layer) proteins are excellent candidates for in vivo and in vitro nanobiotechnological applications because of their ability to self-assemble into two-dimensional lattices that form the outermost layer of many Eubacteria and most Archaea species. Despite this potential, knowledge about their molecular architecture is limited. In this study, we investigated SlpA, the S-layer protein of the potentially probiotic bacterium Lactobacillus brevis ATCC 8287 by cysteine-scanning mutagenesis and chemical modification. We generated a series of 46 mutant proteins by replacing single amino acids with cysteine, which is not present in the wild-type protein. Most of the replaced amino acids were located in the self-assembly domain (residues 179 to 435) that likely faces the outer surface of the lattice. As revealed by electron microscopy, all the mutant proteins were able to form self-assembly products identical to that of the wild type, proving that this replacement does not dramatically alter the protein conformation. The surface accessibility of the sulfhydryl groups introduced was studied with two maleimide-containing marker molecules, TMM(PEG)12 (molecular weight [MW], 2,360) and AlexaFluor488-maleimide (MW = 720), using both monomeric proteins in solution and proteins allowed to self-assemble on cell wall fragments. Using the acquired data and available domain information, we assigned the mutated residues into four groups according to their location in the protein monomer and lattice structure: outer surface of the lattice (9 residues), inner surface of the lattice (9), protein interior (12), and protein-protein interface/pore regions (16). This information is essential, e.g., in the development of therapeutic and other health-related applications of Lactobacillus S-layers.
机译：细菌表面层（S层）蛋白具有自组装成二维晶格的能力，这些蛋白可形成许多 Eubacteria 和大多数细菌的最外层，因此它们是体内和体外纳米生物技术应用的极佳候选者 Archaea 物种。尽管具有这种潜力，但对其分子结构的了解仍然有限。在这项研究中，我们通过半胱氨酸扫描诱变和化学修饰研究了潜在的益生菌“短杆菌” ATCC 8287的S层蛋白SlpA。我们通过用半胱氨酸替换单个氨基酸来生成一系列46个突变蛋白，而半胱氨酸在野生型蛋白中不存在。大多数被替换的氨基酸位于可能面对晶格外表面的自组装域（残基179至435）中。如电子显微镜所揭示的，所有突变蛋白都能够形成与野生型相同的自组装产物，证明这种取代不会显着改变蛋白构象。使用两个含马来酰亚胺的标记分子，TMM（PEG） 12 （分子量[MW]，2,360）和AlexaFluor488-马来酰亚胺（MW = 720），对引入的巯基的表面可及性进行了研究。溶液中的单体蛋白质和蛋白质都可以在细胞壁片段上自组装。使用获得的数据和可用的域信息，我们根据突变的残基在蛋白质单体和晶格结构中的位置将其分为四类：晶格的外表面（9个残基），晶格的内表面（9个），蛋白质内部（12）和蛋白质-蛋白质界面/孔区域（16）。该信息对于例如乳杆菌Sem层的治疗性和其他与健康相关的应用的开发至关重要。

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