Total chemical synthesis of a 27 kDa TASP protein derived from the MscL ion channel of M-tuberculosis by ketoxime-forming ligation
Total chemical synthesis of a 27 kDa TASP protein derived from the MscL ion channel of M-tuberculosis by ketoxime-forming ligation
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DOI：
10.1021/bc010128l
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发表时间：
2002-05-01
影响因子：
4.7
通讯作者：
Cressman, S
中科院分区：
文献类型：
--
作者：
Kochendoerfer, GG;Tack, JM;Cressman, S
通过在携带五个丙酮酸基团的模板分子和携带一个氨氧乙酸基团的线性通道肽之间进行酮肟形成化学选择性连接，合成了源自结核分枝杆菌MscL离子通道的胞质结构域（氨基酸残基103-151）的27-kDa TASP蛋白T(5)Msc(103-151)。酮肟形成连接提供了这种大型全合成蛋白质构建体的高效组装，在氨氧基肽适度过量（1.5x）的情况下，产率>90%。通过 SDS-PAGE 分析和 MALDI 质谱法证实了所需 TASP 分子的形成。通过圆二色光谱评估模板附着对构成 TASP 的肽结构的影响。肽与拓扑模板的连接主要诱导螺旋二级结构，而不带有氨氧基的类似肽 MscL(103-151) 在 pH 7 时不表现出显着的二级结构，并且被发现在浓度高达 65 muM 时呈单体。这一观察结果可以通过 T(5)Msc(103-151) 的展开状态的熵不稳定来解释，这是由于与模板的附着以及由此产生的自由度损失。因此，基于丙酮酸的酮肟形成化学选择性连接可能被证明是组装大型非天然蛋白质构建体及其生物物理研究的有用工具。
A 27-kDa TASP protein, T(5)Msc(103-151), that was derived from the cytoplasmic domain (amino acid residues 103-151) of the MscL ion channel of M. tuberculosis was synthesized by ketoxime-forming chemoselective ligation between a template molecule carrying five pyruvic acid groups, and linear channel peptides carrying one aminooxyacetic acid group. Ketoxime-forming ligation provided for highly efficient assembly of this large totally synthetic protein construct with yields >90% with modest excess (1.5x) of the aminooxy peptide. Formation of the desired TASP molecule was confirmed by SDS-PAGE analysis and MALDI mass spectrometry. The effect of template attachment on the structure of the peptides constituting the TASP was assessed by circular dichroism spectroscopy. Attachment of the peptides to the topological template induces predominantly helical secondary structure, whereas an analogous peptide that did not bear an aminooxy group, MscL(103-151), does not exhibit significant secondary structure at pH 7 and is found to be monomeric in concentrations up to 65 muM. This observation can be explained by entropic destabilization of the unfolded state of T(5)Msc(103-151) due to the attachment to the template and the resulting loss of degrees of freedom. Pyruvic acid-based ketoxime-forming chemoselective ligation may thus prove to be a useful tool for the assembly of large, non-native protein constructs and their biophysical study.