Decreasing the flexibility of the TELSAM-target protein linker and omitting the cleavable fusion tag improves crystal order and diffraction limits.
Decreasing the flexibility of the TELSAM-target protein linker and omitting the cleavable fusion tag improves crystal order and diffraction limits.
复制标题
降低 TELSAM 目标蛋白接头的灵活性并省略可切割的融合标签可改善晶体顺序和衍射极限。
DOI：
10.1101/2023.05.12.540586
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Mood
中科院分区：
文献类型：
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作者：
Gajjar,ParagL;Romo,MariaJPedroza;Litchfield,CelesteM;Callahan,Miles;Redd,Nathan;Nawarathnage,Supeshala;Soleimani,Sara;Averett,Jacob;Wilson,Elijah;Lewis,Andrew;Stewart,Cameron;Tseng,Yi-JieJ;Doukov,Tzanko;Lebedev,Andrey;Mood
TELSAM结晶有望成为蛋白质快速结晶的革命性工具。TELSAM可以提高结晶速率，并在低蛋白质浓度下形成晶体，而无需在TELSAM聚合物之间直接接触，在某些情况下，晶体接触总体上非常小。为了进一步了解和表征TELSAM介导的结晶，我们试图了解TELSAM和融合靶蛋白之间的连接物的组成要求。我们评估了1TEL与人类CMG2 vWa结构域之间的四种不同的连接体Ala-Ala、Ala-Val、Thr-Val和Thr-Thr。我们比较了上述结构的成功结晶条件的数量、晶体数量、平均和最佳衍射分辨率以及细化参数。我们还测试了融合蛋白SUMO对结晶的影响。我们发现连接体的固化提高了衍射分辨率，可能是通过减少晶体中vWa域的可能取向的数量，并且从结构中省略SUMO域也提高了衍射分辨率。我们证明了TELSAM蛋白结晶伴侣可以实现简单的蛋白结晶和高分辨率的结构测定。我们提供的证据支持在TELSAM和感兴趣的蛋白质之间使用短而灵活的连接物，并支持在TELSAM融合构建中避免可切割的纯化标签。
TELSAM crystallization promises to become a revolutionary tool for the facile crystallization of proteins. TELSAM can increase the rate of crystallization and form crystals at low protein concentrations without direct contact between TELSAM polymers and, in some cases, with very minimal crystal contacts overall . To further understand and characterize TELSAM-mediated crystallization, we sought to understand the requirements for the composition of the linker between TELSAM and the fused target protein. We evaluated four different linkers Ala-Ala, Ala-Val, Thr-Val, and Thr-Thr, between 1TEL and the human CMG2 vWa domain. We compared the number of successful crystallization conditions, the number of crystals, the average and best diffraction resolution, and the refinement parameters for the above constructs. We also tested the effect of the fusion protein SUMO on crystallization. We discovered that rigidification of the linker improved diffraction resolution, likely by decreasing the number of possible orientations of the vWa domains in the crystal, and that omitting the SUMO domain from the construct also improved the diffraction resolution.SynopsisWe demonstrate that the TELSAM protein crystallization chaperone can enable facile protein crystallization and high-resolution structure determination. We provide evidence to support the use of short but flexible linkers between TELSAM and the protein of interest and to support the avoidance of cleavable purification tags in TELSAM-fusion constructs.