Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis.
Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis.
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DOI：
10.1083/jcb.109.2.593
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发表时间：
1989-08
期刊：
影响因子：
--
通讯作者：
Weeds AG
中科院分区：
文献类型：
--
作者：
Way M;Gooch J;Pope B;Weeds AG
人血浆凝溶胶蛋白在大肠杆菌中得到了高效可溶性表达。该蛋白具有与血浆凝溶胶蛋白相同的成核和切断活性，并且在与单体肌动蛋白的相互作用中完全对钙敏感。已经表达了许多缺失突变体以探索这三个肌动蛋白结合位点的功能。他们的设计是基于蛋白质序列中的六重片段重复。（这些站点位于段1、段2-3和段4-6）。两个突变体，S1-3和S4-6，相当于通过有限的蛋白水解获得的分子的NH 2-和COOH-末端的一半。S1-3在钙存在或不存在的情况下结合两个肌动蛋白单体，它切断并帽化细丝，但不成核聚合。S4-6结合单个肌动蛋白单体，但仅结合钙离子。这些观察结果证实并扩展了目前对凝溶胶蛋白两半性质的认识。还研究了两种新的构建体，其提供三个位点的不同成对并置。S2-6，其缺少片段1的高亲和力位点（相当于14，000-Mr蛋白水解片段）和S1，4-6，其缺少片段2-3（先前使用28，000-Mr蛋白水解片段鉴定的肌动蛋白丝结合结构域）。S2-6在钙离子中结合两个肌动蛋白单体并使聚合成核;它在钙离子存在或不存在的情况下与细丝横向缔合，并具有弱的钙离子依赖性片段化活性。S1，4-6也结合钙中的两个肌动蛋白单体和EGTA中的一个肌动蛋白单体，具有弱切割活性，但不成核聚合。一个模型的参与的三个结合位点的凝溶胶蛋白的各种活动。
Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.