Hackenberger Christian P R, Friel Claire T, Radford Sheena E, Imperiali Barbara
Department of Chemistry and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Am Chem Soc. 2005 Sep 21;127(37):12882-9. doi: 10.1021/ja051855k.
To establish a system to address questions concerning the influence of glycosylation on protein folding pathways, we have developed a semisynthetic route toward the immunity protein Im7. This fourhelix protein has been used extensively as model protein for folding studies. Native chemical ligation (NCL) affords an N-linked chitobiose glycoprotein analogue of Im7 with an Ala29Cys mutation. The semisynthetic approach relies on the solid-phase peptide synthesis (SPPS) of N-terminal thioesters (including helix I), in glycosylated or unglycosylated form, in combination with the expression of the C-terminal fragment of Im7 (containing helices II-IV). Detailed kinetic and thermodynamic analysis of the protein folding behavior reveals that semisynthetic Im7 analogues are well suited for protein folding studies and that the folding mechanism of the glycoprotein of this Im7 variant is not significantly altered over the unglycosylated analogue.
为建立一个解决糖基化对蛋白质折叠途径影响相关问题的系统，我们开发了一条通往免疫蛋白Im7的半合成路线。这种四螺旋蛋白已被广泛用作折叠研究的模型蛋白。天然化学连接（NCL）可提供具有Ala29Cys突变的Im7的N-连接壳二糖糖蛋白类似物。该半合成方法依赖于以糖基化或非糖基化形式固相合成N端硫酯（包括螺旋I），并结合Im7 C端片段（包含螺旋II-IV）的表达。对蛋白质折叠行为的详细动力学和热力学分析表明，半合成的Im7类似物非常适合蛋白质折叠研究，并且该Im7变体的糖蛋白折叠机制与非糖基化类似物相比没有显著改变。
