Protein encapsulation in the hollow space of hemocyanin crystals containing a covalently conjugated ligand
Protein encapsulation in the hollow space of hemocyanin crystals containing a covalently conjugated ligand
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DOI：
10.1016/j.bbrc.2019.04.062
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发表时间：
2019-06-18
影响因子：
3.1
通讯作者：
Tanaka, Yoshikazu
中科院分区：
文献类型：
--
作者：
Hashimoto, Tsubasa;Ye, Yuxin;Tanaka, Yoshikazu
将客体分子封装到生物大分子晶体的空白空间中已被用于各种目的，包括作为蛋白质容器来保护客体免受物理应力和结构确定。太平洋蟾蜍（Todarodes pacificus）血青素（TpHc）是一种中空的圆柱形十聚体蛋白复合物，其内部空间直径为110 a，高度为160 a。在晶体中，TpHc形成一个类似稻草的束，并在每个原聚体的内部结构域含有一个活性Cys （Cys3246）。在这里，我们将生物素偶联到TpHc的Cys3246上，然后与链霉亲和素孵育。链霉亲和素由于与生物素的相互作用而被固定在TpHc的内部空间。此外，在与未修饰的TpHc相同的条件下，含有TpHc和链霉亲和素的配合物结晶。为了将这种方法扩展到各种蛋白质，我们将与Ni2+离子（Ni2+-NTA）螯合的配体硝基三乙酸（NTA）偶联到TpHc上。我们发现his标记的绿色荧光蛋白（GFP）通过His-tag与Ni2+-NTA基团的相互作用被包被到Ni2+-NTA偶联的TpHc中。x射线晶体学表明，含有TpHc和GFP的配合物的晶体排列与未修饰的TpHc相同。我们的客体固定方法不同于以往依赖客体向宿主晶体扩散的方法。因此，我们的发现可能会加速蛋白质晶体工程的发展。(C) 2019 Elsevier Inc.版权所有。
Encapsulation of guest molecules into the vacant space of biomacromolecular crystals has been utilized for various purposes including functioning as a protein container to protect against physical stress and structural determination of the guest. Todarodes pacificus hemocyanin (TpHc) is a hollow cylindrical decameric protein complex with an inner space 110 A in diameter and 160 A in height. In the crystal, TpHc forms a straw-like bundle and contains one reactive Cys (Cys3246) in the inner domain of each protomer. Here, we conjugated biotin onto Cys3246 of TpHc followed by incubation with streptavidin. The streptavidin was immobilized into the inner space of TpHc due to its interaction with biotin. Moreover, the complex containing TpHc and streptavidin was crystallized under the same conditions used for unmodified TpHc. In order to expand this methodology for a variety of proteins, we conjugated the ligand nitrilotriacetic acid (NTA) chelated to a Ni2+ ion (Ni2+-NTA) to TpHc. We found that His-tagged green fluorescent protein (GFP) was encapsulated into the Ni2+-NTA-conjugated TpHc via the interaction between the His-tag and the Ni2+-NTA group. X-ray crystallography demonstrated that the crystal packing of the complex containing TpHc and GFP was identical to that of the unmodified TpHc. Our guest immobilization method is distinct from previous approaches that are dependent on diffusion of the guest into the host crystal. Thus, our findings may accelerate the development of proteinaceous crystal engineering. (C) 2019 Elsevier Inc. All rights reserved.