SpyAvidin hubs enable precise and ultrastable orthogonal nanoassembly.
SpyAvidin hubs enable precise and ultrastable orthogonal nanoassembly.
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DOI：
10.1021/ja505584f
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发表时间：
2014-09-03
影响因子：
15
通讯作者：
Howarth, Mark
中科院分区：
文献类型：
--
作者：
Fairhead, Michael;Veggiani, Gianluca;Lever, Melissa;Yan, Jun;Mesner, Dejan;Robinson, Carol V.;Dushek, Omer;van der Merwe, P. Anton;Howarth, Mark
链霉亲和素捕获生物素是超分子化学的灵感来源，也是生物化学和纳米技术的核心工具，因为它们之间的相互作用快速而异常稳定。然而，这个中心没有强大的正交相互作用，限制了可以创建的分子组装的大小和复杂性。在这里，我们将traptavidin（链霉亲和素变体最大化生物素结合强度）与正交不可逆相互作用结合。SpyTag是一种经过工程设计的肽，可与其蛋白质伙伴SpyCatcher形成自发的异肽键。SpyTag或SpyCatcher成功融合到死亡链亲和素亚基的c端。我们能够生成具有n（0≤n≤4）个生物素结合位点和4-n个SpyTag或SpyCatcher结合位点的嵌合四聚体。嵌合SpyAvidin四聚体结合精确数量的配体与生物素或SpyTag/SpyCatcher融合。混合嵌合四聚体可以组装SpyAvidin八聚体（8个亚基）或二十聚体（20个亚基）。我们使用电泳和天然质谱法验证了组装。Eicosameric SpyAvidin用于聚类三聚体主要组织相容性复合体（MHC） I类：β2-微球蛋白：肽复合体，产生多达56个组分的组装。MHC二十异构体超越了传统的MHC四聚体，作为一个强大的刺激T细胞信号。结合超稳定的非共价和不可逆的共价相互作用，SpyAvidins可以通过简单的途径创建健壮的纳米结构。
The capture of biotin by streptavidin is an inspiration for supramolecular chemistry and a central tool for biological chemistry and nanotechnology, because of the rapid and exceptionally stable interaction. However, there is no robust orthogonal interaction to this hub, limiting the size and complexity of molecular assemblies that can be created. Here we combined traptavidin (a streptavidin variant maximizing biotin binding strength) with an orthogonal irreversible interaction. SpyTag is a peptide engineered to form a spontaneous isopeptide bond to its protein partner SpyCatcher. SpyTag or SpyCatcher was successfully fused to the C-terminus of Dead streptavidin subunits. We were able to generate chimeric tetramers with n (0 ≤ n ≤ 4) biotin binding sites and 4-n SpyTag or SpyCatcher binding sites. Chimeric SpyAvidin tetramers bound precise numbers of ligands fused to biotin or SpyTag/SpyCatcher. Mixing chimeric tetramers enabled assembly of SpyAvidin octamers (8 subunits) or eicosamers (20 subunits). We validated assemblies using electrophoresis and native mass spectrometry. Eicosameric SpyAvidin was used to cluster trimeric major histocompatibility complex (MHC) class I:β2-microglobulin:peptide complexes, generating an assembly with up to 56 components. MHC eicosamers surpassed the conventional MHC tetramers in acting as a powerful stimulus to T cell signaling. Combining ultrastable noncovalent with irreversible covalent interaction, SpyAvidins enable a simple route to create robust nanoarchitectures.
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