Disulfide mapping of the cyclotide kalata B1 -: Chemical proof of the cyclic cystine knot motif
Disulfide mapping of the cyclotide kalata B1 -: Chemical proof of the cyclic cystine knot motif
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DOI：
10.1074/jbc.m308771200
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发表时间：
2003-11-28
影响因子：
4.8
通讯作者：
Craik, DJ
中科院分区：
文献类型：
--
作者：
Göransson, U;Craik, DJ
环肽是最近发现的一类植物蛋白，具有连续的环状骨架和三个保守的二硫键的打结排列的迷人结构特征。我们在这里给出了典型的环肽Kalata B1的I-IV，II-V，III-VI打结的二硫键连接性的明确的化学证据。这是通过一种新的二硫化物分析方法实现的，包括部分还原和逐步烷基化，包括通过半胱氨酸的氨乙基化引入电荷和酶切位点。该方法克服了环肽二硫键作图的固有困难，即环酰胺主链、半胱氨酸之间缺乏裂解中心以及碱性氨基酸含量低或聚集，并允许使用质谱分析直接确定Kalata B1中的二硫键。通过拓扑分析，已建立的二硫键连接性明确地显示为胱氨酸打结。这是首次直接测定天然环类化合物中二硫化物的含量，并明确证实了独特的环状胱氨酸结基序。
The cyclotides are a recently discovered family of plant proteins that have the fascinating structural feature of a continuous cyclic backbone and, putatively, a knotted arrangement of their three conserved disulfide bonds. We here show definite chemical proof of the I-IV, II-V, III-VI knotted disulfide connectivity of the prototypic cyclotide kalata B1. This has been achieved by a new approach for disulfide analysis, involving partial reduction and stepwise alkylation including introduction of charges and enzymatic cleavage sites by aminoethylation of cysteines. The approach overcomes the intrinsic difficulties for disulfide mapping of cyclotides, i.e. the cyclic amide backbone, lack of cleavage sites between cysteines, and a low or clustered content of basic amino acids, and allowed a direct determination of the disulfide bonds in kalata B1 using analysis by mass spectrometry. The established disulfide connectivity is unequivocally shown to be cystine knotted by a topological analysis. This is the first direct chemical determination of disulfides in native cyclotides and unambiguously confirms the unique cyclic cystine knot motif.