The structural basis of nanobody unfolding reversibility and thermoresistance.
The structural basis of nanobody unfolding reversibility and thermoresistance.
复制标题
DOI：
10.1038/s41598-018-26338-z
复制
发表时间：
2018-05-21
影响因子：
4.6
通讯作者：
Hoheisel JD
中科院分区：
文献类型：
--
作者：
Kunz P;Zinner K;Mücke N;Bartoschik T;Muyldermans S;Hoheisel JD
纳米抗体代表骆驼科重链抗体的可变结合结构域，并且在生物技术和生物医学中的应用范围迅速增长。他们的成功是基于独特的性质，包括他们的报告能力，可逆地重折叠后热诱导变性。然而，这种观点与涉及不可逆聚集纳米抗体的研究形成对比，要求进行定量分析，明确定义纳米抗体的耐热性，并揭示解折叠可逆性和聚集倾向的决定因素。通过表征近70种纳米抗体，我们表明，对于大多数结合剂，在热变性时确实会发生不可逆的聚集，这可能会影响稳定性和免疫原性等应用相关参数。然而，通过从表观熔化温度导出聚集倾向，我们表明可选的二硫键抑制纳米抗体聚集。通过增加互补决定环的长度进一步增强这种效果，尽管预期互补决定环会使纳米抗体不稳定，但有助于纳米抗体稳定性。然而，这种变化的影响取决于环境条件。例如，具有两个二硫键的纳米抗体易于在胞质溶胶中失去其功能性。我们的研究提出了设计纳米抗体的策略，这些纳米抗体表现出最佳的性能参数，并深入了解了进化以防止蛋白质聚集的一般机制。
Nanobodies represent the variable binding domain of camelid heavy-chain antibodies and are employed in a rapidly growing range of applications in biotechnology and biomedicine. Their success is based on unique properties including their reported ability to reversibly refold after heat-induced denaturation. This view, however, is contrasted by studies which involve irreversibly aggregating nanobodies, asking for a quantitative analysis that clearly defines nanobody thermoresistance and reveals the determinants of unfolding reversibility and aggregation propensity. By characterizing nearly 70 nanobodies, we show that irreversible aggregation does occur upon heat denaturation for the large majority of binders, potentially affecting application-relevant parameters like stability and immunogenicity. However, by deriving aggregation propensities from apparent melting temperatures, we show that an optional disulfide bond suppresses nanobody aggregation. This effect is further enhanced by increasing the length of a complementarity determining loop which, although expected to destabilize, contributes to nanobody stability. The effect of such variations depends on environmental conditions, however. Nanobodies with two disulfide bonds, for example, are prone to lose their functionality in the cytosol. Our study suggests strategies to engineer nanobodies that exhibit optimal performance parameters and gives insights into general mechanisms which evolved to prevent protein aggregation.
登录
查看更多内容
影响因子：
4.6
作者：
Beghein E;Van Audenhove I;Zwaenepoel O;Verhelle A;De Ganck A;Gettemans J
通讯作者：
Gettemans J
影响因子：
7.3
作者：
Goldman ER;Liu JL;Zabetakis D;Anderson GP
通讯作者：
Anderson GP
影响因子：
4.8
作者：
Akazawa-Ogawa, Yoko;Takashima, Mizuki;Hagihara, Yoshihisa
通讯作者：
Hagihara, Yoshihisa
影响因子：
4.8
作者：
Govaert, Jochen;Pellis, Mireille;Saerens, Dirk
通讯作者：
Saerens, Dirk
影响因子：
8
作者：
Dumoulin, M;Conrath, K;Matagne, A
通讯作者：
Matagne, A