Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot
Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot
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DOI：
10.1021/bi049711q
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发表时间：
2004-05-25
期刊：
影响因子：
2.9
通讯作者：
Craik, DJ
中科院分区：
文献类型：
--
作者：
Colgrave, ML;Craik, DJ
环肽是最近发现的一类植物源肽，具有首尾相连的环化骨架和胱氨酸结核心的不寻常特征。这些特征被认为有助于它们的特殊稳定性，正如在旨在测序和表征该家族早期成员的实验中定性观察到的那样。然而，迄今为止，还没有定量研究的热，化学，或酶的稳定性的环肽。在这项研究中，我们证明了原型cyclotide kalata B1的稳定性的离液剂6 M盐酸胍（GdHCl）和8 M尿素，温度接近沸腾，酸，并与一系列的蛋白酶，条件下，大多数蛋白质容易展开孵育。NMR光谱用于证明热稳定性，而荧光和圆二色性用于监测化学稳定性。还检查了几种kalata B1的变体，包括kalata 132，其具有来自B1的五个氨基酸取代，两个无环排列体，其中主链断裂但保留胱氨酸结，以及两个二硫键突变体。总之，这些可以确定胱氨酸结和环状骨架对环肽稳定性的相对作用。添加变性剂的kalata B1或无环排列没有引起展开，但两个二硫化物衍生物是不太稳定的，尽管具有类似的三维结构。这表明，胱氨酸结在环肽的化学稳定性方面比环状骨架更重要。此外，通过与芋螺毒素PVIIA的比较判断，环肽的胱氨酸结比类似大小的分子中的胱氨酸结更稳定。通过LC-MS监测，没有天然kalata B1酶消化的证据，但还原形式对胰蛋白酶、内切蛋白酶Glu-C和嗜热菌蛋白酶的蛋白水解敏感。在还原剂二硫苏糖醇的存在下，kalata B1的荧光光谱显示出强度的显著增加，这被认为是由于相邻的Cys 5-Cys 17二硫键去除了对Trp残基的淬灭作用。一般来说，还原肽比氧化物质更容易发生化学或酶促分解。
The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata 132, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.