Backbone Circularization Coupled with Optimization of Connecting Segment in Effectively Improving the Stability of Granulocyte-Colony Stimulating Factor
Backbone Circularization Coupled with Optimization of Connecting Segment in Effectively Improving the Stability of Granulocyte-Colony Stimulating Factor
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DOI：
10.1021/acschembio.7b00776
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发表时间：
2017-10-01
影响因子：
4
通讯作者：
Honda, Shinya
中科院分区：
文献类型：
--
作者：
Miyafusa, Takamitsu;Shibuya, Risa;Honda, Shinya
蛋白质骨架环化是提高蛋白质结构稳定性的有效方法。在本文中，我们假设紧密连接导致更高的稳定性。因此，我们设计了具有结构优化的末端连接的粒细胞集落刺激因子（G-CSF）的环化变体。为了估计连接的适当长度，我们调查了蛋白质数据库，以找到局部结构作为连接片段的模型。我们建立了由“螺旋-环-螺旋”组成的局部结构库，随后选择与G-CSF末端相似的条目，最后根据环长度对命中结构进行排序。经常观察到两个、五个或九个环残基;因此，构建、制备和评价了三个环化变体（C163、C166和C170）。所有环化变体均表现出比线性G-CSF（L175）更高的热稳定性。特别地，保留5个连接残基的C166表现出69.4 ° C的表观T-m值，其比没有截断的环化变体（C177）高8.7 ° C，表明连接片段的优化对于增强整体结构稳定性是有效的。C166还显示出比L175更高的对内切蛋白酶和外肽酶的蛋白水解稳定性。本研究为环化蛋白的设计和G-CSF生物优化剂的开发提供了理论依据。
Backbone circularization of protein is a powerful method to improve its structural stability. In this paper, we presumed that a tight connection leads to much higher stability. Therefore, we designed circularized variants of a granulocyte-colony stimulating factor (G-CSF) with a structurally optimized terminal connection. To estimate the appropriate length of the connection, we surveyed the Protein Data Bank to find local structures as a model for the connecting segment. We set the library of local structures composed of "helix-loop-helix," subsequently selected entries similar to the G-CSF terminus, and finally sorted the hit structures according to the loop length. Two, five, or nine loop residues were frequently observed; thus, three circularized variants (C163, C166, and C170) were constructed, prepared, and evaluated. All circularized variants demonstrated a higher thermal stability than linear G-CSF (L175). In particular, C166 that retained five connecting residues demonstrated apparent T-m values of 69.4 degrees C, which is 8.7 degrees C higher than that of the circularized variant with no truncation (C177), indicating that the optimization of the connecting segment is effective for enhancing the overall structural stability. C166 also showed higher proteolytic stability against both endoprotease and exopeptidase than L175. We anticipate that the present study will contribute to the improvement in the general design of circularized protein and development of G-CSF biobetters.