Exploiting the substrate tolerance of farnesyltransferase for site-selective protein derivatization
Exploiting the substrate tolerance of farnesyltransferase for site-selective protein derivatization
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DOI：
10.1002/cbic.200600440
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发表时间：
2007-03-05
期刊：
影响因子：
3.2
通讯作者：
Waldmann, Herbert
中科院分区：
文献类型：
--
作者：
Nguyen, Uyen T. T.;Cramer, Janina;Waldmann, Herbert
蛋白质的功能基团定点修饰是一种重要的生化技术，但其他氨基酸侧链的反应使所需基团与所选位点的共价连接变得复杂，经常导致不希望发生的副反应。这个问题的一个潜在解决方案是利用蛋白质修饰酶的活性，这种酶可以识别特定的蛋白质序列。蛋白法尼基转移酶(FTase)在一个短的C末端序列的背景下，将一个类异戊二烯部分共价连接到半胱氨酸单元上，该序列可以很容易地嫁接到重组蛋白上。在这里，我们描述了用生物素、叠氮或二烯基团官能化的四个磷酸异戊二烯类化合物的合成。这些磷酸异戊二烯类化合物与FTase结合，亲和力与天然底物相当。除了生物素功能化的类似物外，所有生成的磷异戊二烯类化合物都可以通过FTase转移到多肽和蛋白质底物上。与法尼基修饰的蛋白质不同，用(2E，6E)-8-(azidoacetamido)-3，7-dimethylocta-2，6-dienyl基团修饰的YPT7不会齐聚，也没有表现出明显的疏水性增加。为了评估功能化的异戊二烯类化合物对蛋白质修饰的适用性，通过饮食-Alder与6-马来亚胺基己酸的环加成反应和通过Staudinger与磷化氢的连接来进一步衍生化。我们证明了Staudinger配位反应比Diels-Alder环加成反应进行得更快、更有效。我们的数据验证了FTase作为生化和生物技术应用中的蛋白质修饰工具的使用。
The site-selective modification of proteins with a functional group is an important biochemical technique, but covalent attachment of a desired group to a chosen site is complicated by the reactivity of other amino acid side chains, often resulting in undesired side reactions. One potential solution to this problem involves exploiting the activity of protein-modifying enzymes that recognize a defined protein sequence. Protein farnesyltransferase (FTase) covalently attaches an isoprenoid moiety to a cysteine unit in the context of a short C-terminal sequence that can be easily grafted onto recombinant proteins. Here we describe the synthesis of four phosphoisoprenoids functionalized with biotin, azide, or diene groups. These phosphoisoprenoids bound to FTase with affinities comparable to that of the native substrate. with the exception of the biotin-functionalized analogue, all the phos-phoisoprenoids generated could be transferred to peptide and protein substrates by FTase. Unlike proteins modified with farnesyl moieties, Ypt7 prenylated with (2E,6E)-8-(azidoacetamido)-3,7-dimethylocta-2,6-dienyl groups did not oligomerize and showed no detectable increase in hydrophobicity. To assess the suitability of the functionalized isoprenoids for protein modifications they were further derivatized, both by Diets-Alder cycloaddition with 6-maleimidohexanoic acid and by Staudinger ligation with a phosphine. We demonstrate that the Staudinger ligation proceeds more rapidly and is more efficient than the Diels-Alder cycloaddition. Our data validate the use of FTase as a protein-modification tool for biochemical and biotechnological applications.