Functional characterization and NMR Spectroscopy on full-length Vpu from HIV-1 prepared by total chemical synthesis
Functional characterization and NMR Spectroscopy on full-length Vpu from HIV-1 prepared by total chemical synthesis
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DOI：
10.1021/ja038985i
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发表时间：
2004-03-03
影响因子：
15
通讯作者：
Montal, M
中科院分区：
文献类型：
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作者：
Kochendoerfer, GG;Jones, DH;Montal, M
Vpu是HIV-1基因组中编码的81个残基的完整膜蛋白，由于它在病毒颗粒从感染细胞释放和细胞受体降解中起重要作用，因此引起了相当大的兴趣。我们在此报道了全长Vpu（1-81）和一个位点特异性的n -15标记类似物Vpu（2-81）的全化学合成，使用天然化学连接方法，并报道了这些构建物与通过细菌表达获得的重组蛋白的结构和功能比较。合成的多肽和表达的多肽在脂质胶束中的结构相似。排列的水合脂双分子层中多肽的固体核磁共振光谱表明，它们的整体拓扑结构也非常相似。此外，发现合成蛋白的通道活性类似于先前表征的重组蛋白。因此，我们已经证明，使用固相肽合成和化学连接是可行的，以获得大量的纯化和均匀的膜蛋白，其结构和功能相关的形式，为未来的结构和表征研究。
Vpu is an 81-residue integral membrane protein encoded in the HIV-1 genome that is of considerable interest because it plays important roles in the release of virus particles from infected cells and in the degradation of the cellular receptor. We report here the total chemical synthesis of full-length Vpu(1-81) as well as a site-specifically N-15-labeled analogue, Vpu(2-81), using native chemical ligation methodologies and also report a structural and functional comparison of these constructs with recombinant protein obtained via bacterial expression. The structures of the synthetic and expressed polypeptides were similar in lipid micelles using solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in aligned hydrated lipid bilayers indicated that their overall topologies were also very comparable. Further, the channel activity of the synthetic protein was found to be analogous to that previously characterized for the recombinant protein. We have thus demonstrated that using solid phase peptide synthesis and chemical ligation it is feasible to obtain large quantities of a purified and homogeneous membrane protein in a structurally and functionally relevant form for future structural and characterization studies.