Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, C/Fuentenueva s/n, Granada, Spain.
Biochimie. 2011 Mar;93(3):549-55. doi: 10.1016/j.biochi.2010.11.011. Epub 2010 Dec 2.
To confirm whether the head-to-tail circularization could be involved in the stability and activity of the circular bacteriocin AS-48, two permutated linear structural as-48A genes have been constructed by circular permutation. The absence of the leaderless linear AS(23/24) and AS(48/49) proteins in Escherichia coli, under all the conditions investigated, supports the idea that the circular backbone is important to stabilize their structure and also indicates the significance of a leader peptide. In fact, the approach taken in this study to generate linear permutated proteins fused to an appropriate partner was sufficient to prevent cellular proteolysis. In this case, the high expression levels found favour their intracellular accumulations as inclusion bodies, which after solubilization showed a propensity to aggregate, thus hindering the specific EK cleavage. This could explain the presence of active hybrid tagged proteins identified in this work. The conserved distribution of hydrophobic and hydrophilic surfaces in the hybrid proteins is responsible for the antibacterial activity. In addition, the opening of the AS-48 molecule between the residues G(23) W(24) connecting the α1/α2 helices, confers greater stability, suggesting that the sequence and/or the free amino acid in the polypeptide chain are critical aspects in the design of new variants.
为了确认头到尾的环状化是否参与了环状细菌素 AS-48 的稳定性和活性，通过环状移位构建了两个移位的线性结构 as-48A 基因。在所有研究的条件下，大肠杆菌中没有无领袖的线性 AS(23/24)和 AS(48/49)蛋白，这支持了环状骨架对稳定其结构很重要的观点，也表明了前导肽的重要性。事实上，本研究中采用的生成融合到适当伴侣的线性移位蛋白的方法足以防止细胞蛋白水解。在这种情况下，发现的高表达水平有利于它们作为包含体的细胞内积累，这些包含体在溶解后表现出聚集的倾向，从而阻碍了特定的 EK 切割。这可以解释在这项工作中发现的有活性的杂交标记蛋白的存在。在杂交蛋白中疏水和亲水表面的保守分布负责其抗菌活性。此外，在连接 α1/α2 螺旋的残基 G(23)W(24)之间的 AS-48 分子的开口赋予了更大的稳定性，这表明在多肽链中的序列和/或游离氨基酸是设计新变体的关键方面。
