Isolation and characterization of novel cyclotides from Viola hederaceae -: Solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide
Isolation and characterization of novel cyclotides from Viola hederaceae -: Solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide
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DOI：
10.1074/jbc.m501737200
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发表时间：
2005-06-10
影响因子：
4.8
通讯作者：
Craik, DJ
中科院分区：
文献类型：
--
作者：
Chen, B;Colgrave, ML;Craik, DJ
基于新建立的高效、简单、易操作的测序策略，对从澳大利亚植物堇菜常春藤中分离的四种新型环肽（大环打结蛋白）进行了序列测定。 V. hederaceae 叶环肽-1 ( vhl-1) 是一种叶特异性表达的 31 个残基环肽，已使用二维 H-1 NMR 光谱测定了其三维溶液结构。 vhl-1采用紧凑且明确的结构，包括扭曲的三链β折叠、短的310螺旋段和多个转角。它由三个二硫键稳定，与主链片段一起形成环状胱氨酸结基序。三个二硫键几乎完全埋入蛋白质核心，六个半胱氨酸仅占分子表面的3.8%。 pH 滴定实验表明，vhl-1 的折叠显示出很少的 pH 依赖性，并且可以确定 Glu(3) 的 pK(a) 为 3.0，Glu(14) 的 pK(a) 类似于 5.0。发现 Met(7) 在天然形式下被氧化，这与其侧链伸入溶剂中、占据分子表面的 7.5% 的事实相一致。 vhl-1显示出抗HIV活性，EC50值为0.87μm。
Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 ( vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional H-1 NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded beta- sheet, a short 310 helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pK(a) of 3.0 for Glu(3) and similar to 5.0 for Glu(14) to be determined. Met(7) was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC50 value of 0.87 mu m.