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首页> 外文期刊>Chemistry: A European journal >Synthesis of Functionalized Rab GTPases by a Combination of Solution-or Solid-Phase Lipopeptide Synthesis with Expressed Protein Ligation
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Synthesis of Functionalized Rab GTPases by a Combination of Solution-or Solid-Phase Lipopeptide Synthesis with Expressed Protein Ligation

机译：溶液或固相脂肽合成与表达蛋白连接的合成合成功能化Rab GTPases。

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Prenylated proteins with non-natIVe functionalities are generally very difficult to obtain by recombinant or enzymatic means.The semisynthesis of preparatIVe amounts of prenylated Rab guanosine triphosphatases(GTPases)from recombinant proteins and synthetic prenylated peptides depends largely on the availability of functionalised prenylated peptides corresponding to the proteins' natIVe structure or modifications thereof.Here,we describe and compare solution-phase and solid-phase strategies for the generation of peptides corresponding to the prenylated C terminus of Rab7 GTPase.The solid-phase with utilisation of a hydrazide linker emerges as the more favourable approach.It allows a fast and practical synthesis of pure peptides and gIVes a high degree of flexibility in their modification.To facilitate the analysis of semisynthetic proteins,the synthesised peptides were equipped with a fluorescent group.Using the described approach,we introduced fluorophores at several different positions of the Rab7 C terminus.The position of the incorporated fluorescent groups in the peptides did not influence the protein-ligation reaction,as the generated peptides could be li-gated onto thioester-tagged Rab7.However,it was found that the positioning of the fluorescent group had an influence on the functionality of the Rab7 proteins;analysis of the interaction of the semisynthetic Rab7 proteins with REP(Rab escort protein)and GDI(guanosine diphosphate dissociation inhibitor)molecules revealed that modification of the peptide side chains or of the C-terminal isoprenoid did not significantly interfere with complex formation.However,functionalisation of the C terminus was found to have an adverse effect on complex formation and stability,possibly reflecting low structural flexibility of the Rab GDI/ REP molecules in the vicinity of the lipid-binding site.
机译：通常很难通过重组或酶促方法获得具有非natIVe功能的异戊二烯基化蛋白。重组蛋白和合成的异戊二烯基化肽半合成的异戊二烯基化的Rab鸟苷三磷酸酶（GTPases）的半合成量很大程度上取决于官能化的异戊二烯化肽的可用性在此，我们描述并比较了溶液相和固相策略，用于生成与Rab7 GTPase的烯丙基化C端相对应的肽。利用酰肼连接基的固相出现如下：它允许快速，实用地合成纯肽和gIVes，并具有高度的修饰灵活性。为方便半合成蛋白的分析，合成的肽配备了荧光基团。在几个不同的位置引入荧光团肽中掺入的荧光基团的位置不影响蛋白质的连接反应，因为生成的肽可以连接到硫代酯标记的Rab7上。但是，发现荧光基团对Rab7蛋白质的功能有影响;半合成Rab7蛋白质与REP（Rab伴游蛋白）和GDI（鸟苷二磷酸解离抑制剂）分子的相互作用分析表明，肽侧链或C末端类异戊二烯没有显着干扰复合物的形成。但是，发现C末端的功能化对复合物的形成和稳定性有不利影响，可能反映了Rab GDI / REP分子在脂质附近的结构柔性低。 -结合位点。

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