Plug-and-play pairing via defined divalent streptavidins.
Plug-and-play pairing via defined divalent streptavidins.
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DOI：
10.1016/j.jmb.2013.09.016
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发表时间：
2014-01-09
影响因子：
5.6
通讯作者：
Howarth, Mark
中科院分区：
文献类型：
--
作者：
Fairhead, Michael;Krndija, Denis;Lowe, Ed D.;Howarth, Mark
链霉亲和素是分子生物学中最重要的中心之一，它可以聚合生物分子，桥接一个分子到另一个分子，或者锚定在生物素化的表面/纳米颗粒上。链霉亲和素具有与生物素快速超稳定结合的优点。然而，链霉亲和素以异质方式结合四种生物素化分子的能力往往是有限的。在这里，我们提出了一种有效的方法来分离具有两个生物素结合位点的链霉亲和素四聚体，其精确排列为顺式或反式。我们对带负电荷标签的特定亚基进行了基因修饰，对单体混合物进行了折叠，并根据标签的数量和取向使用离子交换色谱法来分离四聚体。我们将顺式二价链霉亲和素的晶体结构求解为1.4 Å分辨率，反式二价链霉亲和素的晶体结构求解为1.6 Å分辨率，验证了分离策略并解释了死亡链霉亲和素变体的行为。顺式和反式二价链霉亲和素保留了四价链霉亲和素的高热稳定性和低脱除率。这些确定的二价链霉亲和素使我们能够揭示链霉亲和素的结合如何取决于生物素配体的性质。生物素化的DNA对顺二价链霉亲和素表现出很强的负协同性，而对反二价链霉亲和素则没有。易与顺式和反式结合位点结合的生物素化的小蛋白质。我们还解决了与生物素-4-荧光素结合的反式二价链亲和素的结构，显示了一个配体如何阻碍与邻近生物素结合位点的结合。使用六甲酰胺标签被证明是一种更有效的分离单价链霉亲和素的方法，具有超稳定的标记，没有不必要的聚类。这些形式的链亲和素允许这个关键枢纽被用于一个新的精度水平，均匀的分子组装。链霉亲和素是一种广泛使用的纳米中心：通常，四价使得组装不精确。我们分离出具有顺式或反式生物素结合位点的二价链霉亲和素。顺式和反式二价链亲和素保留了特殊的生物素结合和热稳定性。将大的生物素化配体与邻近的结合位点结合是非常不利的。顺式和反式二价链亲和素是一种精确桥接生物分子的简单方法。
Streptavidin is one of the most important hubs for molecular biology, either multimerizing biomolecules, bridging one molecule to another, or anchoring to a biotinylated surface/nanoparticle. Streptavidin has the advantage of rapid ultra-stable binding to biotin. However, the ability of streptavidin to bind four biotinylated molecules in a heterogeneous manner is often limiting. Here, we present an efficient approach to isolate streptavidin tetramers with two biotin-binding sites in a precise arrangement, cis or trans. We genetically modified specific subunits with negatively charged tags, refolded a mixture of monomers, and used ion-exchange chromatography to resolve tetramers according to the number and orientation of tags. We solved the crystal structures of cis-divalent streptavidin to 1.4 Å resolution and trans-divalent streptavidin to 1.6 Å resolution, validating the isolation strategy and explaining the behavior of the Dead streptavidin variant. cis- and trans-divalent streptavidins retained tetravalent streptavidin's high thermostability and low off-rate. These defined divalent streptavidins enabled us to uncover how streptavidin binding depends on the nature of the biotin ligand. Biotinylated DNA showed strong negative cooperativity of binding to cis-divalent but not trans-divalent streptavidin. A small biotinylated protein bound readily to cis and trans binding sites. We also solved the structure of trans-divalent streptavidin bound to biotin-4-fluorescein, showing how one ligand obstructs binding to an adjacent biotin-binding site. Using a hexaglutamate tag proved a more powerful way to isolate monovalent streptavidin, for ultra-stable labeling without undesired clustering. These forms of streptavidin allow this key hub to be used with a new level of precision, for homogeneous molecular assembly. Streptavidin is a widely used nanohub: often, tetravalency makes assembly imprecise. We isolated divalent streptavidins with biotin-binding sites in cis or in trans. cis- and trans-divalent streptavidins retain exceptional biotin binding and thermostability. Binding large biotinylated ligands to adjacent binding sites is greatly disfavored. cis- and trans-divalent streptavidins represent a simple way to bridge biomolecules precisely.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
15
作者：
Blois, Tracy M.;Hong, Heedeok;Bowie, James U.
通讯作者：
Bowie, James U.
影响因子：
48
作者：
Howarth, M;Chinnapen, DJF;Ting, AY
通讯作者：
Ting, AY
影响因子：
2.7
作者：
Funabashi, Hisakage;Ubukata, Michito;Kobatake, Eiry
通讯作者：
Kobatake, Eiry
影响因子：
4.1
作者：
GREEN, NM
通讯作者：
GREEN, NM