IDENTIFICATION OF DISULFIDE-BRIDGED SUBSTRUCTURES WITHIN HUMAN VONWILLEBRAND-FACTOR
IDENTIFICATION OF DISULFIDE-BRIDGED SUBSTRUCTURES WITHIN HUMAN VONWILLEBRAND-FACTOR
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DOI：
10.1021/bi00399a013
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发表时间：
1987-12-15
期刊：
影响因子：
2.9
通讯作者：
WALSH, KA
中科院分区：
文献类型：
--
作者：
MARTI, T;ROSSELET, SJ;WALSH, KA
在鉴定同源低聚蛋白von Willebrand因子[每个多肽链270千道尔顿(KDa)]的亚结构域的过程中，有限的蛋白质降解产生了7个8-90 kDa的大片段。与凝血因子VIIIc结合的单体片段被鉴定为残基1-272。结合血小板糖蛋白Ib的片段被鉴定为含有两对相同链的同源二聚体，即残基273-511和674-728。已有几种方法鉴定了二硫键，包括直接观察半胱氨酸在Edman降解过程中的苯硫代海因。在氨基末端1365个残基的半胱氨酸残基中，有52个已配对。它们在三个结构域内对折叠的可能性施加结构约束。另外的二硫键簇也很明显。研究表明，至少有35个二硫键必须形成链内桥，特别是残基1-272和906-1492之间的半胱氨酸。亚基间二硫键部分定位在内部区域(残基283-695)和羧基末端区域(残基1908-2050)。这些区域中的每一个似乎都链接到互连链网络中相邻亚单位的相应区域。评估了所有169个半胱氨酸(每条链)配对以及区分链间、链间和链间二硫化物的困难。
In the course of identifying substructural domains within the homooligomeric protein von Willebrand factor [270 kilodaltons (kDa) per polypeptide chain], seven large fragments of 8-90 kDa have been generated by limited proteolysis. A monomeric fragment that binds coagulation factor VIIIc is identified as residues 1-272. A fragment that binds platelet glycoprotein Ib is identified as a homodimer containing two pairs of identical chains, i.e., residues 273-511 and 674-728. Disulfide bonds have been identified by several methods, including direct observation of the phenylthiohydantoin of cysteine during Edman degradation of isolated peptides. Among half-cystine residues in the amino-terminal 1365-residue region, 52 have been paired. They place structural constraints on folding possibilities within three structural domains. Additional clusters of disulfide bonds are evident. It has been shown that at least 35 disulfides must form intrachain bridges, specially the cystines among residues 1-272 and 906-1492. Intersubunit disulfide bonds are partially localized in an interiorregion (residues 283-695) and a carboxyl-terminal regions (residues 1908-2050). Each of these regions appears to be linked to a corresponding region of a neighboring subunit in the network of interconnected chains. The difficulties of pairing all 169 half-cystines (per chain) and of distinguishing interchain from interchain from interchain disulfides are evaluated.