Structure-function studies of the plant cyclotides: The role of a circular protein backbone
Structure-function studies of the plant cyclotides: The role of a circular protein backbone
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DOI：
10.1081/txr-120026914
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发表时间：
2003-01-01
期刊：
影响因子：
--
通讯作者：
Sando, L
中科院分区：
文献类型：
--
作者：
Craik, DJ;Barry, DG;Sando, L
采用毛细管电泳法，对大肠杆菌进行单酶分子检测。大肠杆菌β-半乳糖苷酶从三个不同的样品集。第一组由来自五种不同细菌菌株的诱导细胞的裂解物以及两种不同的酶的商业制剂组成。发现这些样品具有显著不同的单分子活性分布。对于第二组样品，诱导β-半乳糖苷酶表达1.5小时，然后进一步孵育，其中表达被抑制。对诱导前的裂解物和诱导后设定时间取的等分试样的裂解物进行测定。最近诱导的酶具有比基础表达的酶高25%的平均单分子活性。这一平均活性在诱导后3.5小时恢复到基础值，此后保持不变。最后，在26和42 ℃下诱导β-半乳糖苷酶。在部分热变性之前和之后测定酶。这些样品被发现在它们的平均单分子活性方面是不可区分的。传统的蛋白质是线性氨基酸链的想法受到了挑战，发现了含有环状骨架的小蛋白。环肽家族是环状蛋白中最大的一组，其特征在于酰胺环化的蛋白质骨架和六个保守的半胱氨酸残基。这些保守的半胱氨酸配对形成二硫键的打结网络。环状骨架和胱氨酸结的组合，称为环状胱氨酸结（CCK）基序，赋予环肽特殊的稳定性。这篇综述讨论的基础上的氧化折叠的kalata B1，原型cyclotide，和比较的结构和活性的kalata B1和它的无环排列的研究环状骨架的作用。
Using a capillary electrophoresis-based method, single enzyme molecule assays were performed on E. coli beta-galactosidase from three different sets of samples. The first set consisted of lysates of induced cells from five different strains of the bacteria, as well as two different commercial preparations of the enzyme. These samples were found to have substantially different distributions of single molecule activities. For the second set of samples, beta-galactosidase expression was induced for 1.5 hr, followed by further incubation where expression was repressed. Assays were performed on the lysates of the preinduction and on the lysates from aliquots taken set times postinduction. The recently induced enzyme had a 25% higher average single molecule activity than the basally expressed enzyme. This average activity returned to the basal value 3.5 hr postinduction and remained unchanged thereafter. Finally, beta-galactosidase was induced at 26 and 42degreesC. The enzyme was assayed before and after partial thermal denaturation. The samples were found to be indistinguishable with respect to their average single molecule activities.The traditional idea of proteins as linear chains of amino acids is being challenged with the discovery of miniproteins that contain a circular backbone. The cyclotide family is the largest group of circular proteins and is characterized by an amide-circularized protein backbone and six conserved cysteine residues. These conserved cysteines are paired to form a knotted network of disulfide bonds. The combination of the circular backbone and a cystine knot, known as the cyclic cystine knot (CCK) motif, confers exceptional stability upon the cyclotides. This review discusses the role of the circular backbone based on studies of both the oxidative folding of kalata B1, the prototypical cyclotide, and a comparison of the structure and activity of kalata B1 and its acyclic permutants.