Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 20, 14195 Berlin, Germany.
Biomacromolecules. 2023 Jul 10;24(7):3357-3369. doi: 10.1021/acs.biomac.3c00427. Epub 2023 Jun 28.
The α-helical coiled coil (CC) is one of the best-characterized folding motifs in the protein world. In this context, fluorinated amino acids have been shown to be capable of tuning the properties of CC assemblies, and especially fluorinated derivatives of aliphatic amino acids can significantly increase the stability of this folding motif when placed in the hydrophobic and positions. However, it has not been shown yet whether fluorinated amino acids, by means of rational design, can be used as an orthogonal tool to control CC assembly processes. In the current work, we approached this question by creating a combinatorial peptide library based on a VPE/VPK heteromeric CC system previously established and characterized in our group. This CC model allowed us to screen fluorinated amino acids for interaction with different potential binding partners in position of the VPE/VPK model with a particular emphasis on studying the impact of stereochemistry within the side chain of α-branched aliphatic fluorinated amino acids on CC properties such as oligomerization state, thermodynamic stability, and orientation. 28 combinations of library members were characterized regarding structure, oligomerization, and thermal stability utilizing circular dichroism, size exclusion chromatography, and Förster resonance energy transfer measurements. This detailed approach showed that the stability and oligomerization state of the motif were not only dependent on the steric demand and the fluorination of corresponding amino acids but also on the stereochemistry within the side chain. The results were applied for a rational design of the fluorine-driven orthogonal assembly, and we could show that CC dimer formation occurred based on specific interactions between fluorinated amino acids. These results demonstrate the potential of fluorinated amino acids as an orthogonal tool besides classical electrostatic and hydrophobic interactions for the fine-tuning and direction of peptide-peptide interactions. Furthermore, within the space of fluorinated amino acids, we could demonstrate the specificity of interactions between differently fluorinated side chains.
α-螺旋卷曲螺旋（CC）是蛋白质世界中研究最为透彻的折叠基序之一。在这种情况下，已经证明氟化氨基酸能够调节 CC 组装体的性质，并且当将氟化的脂肪族氨基酸衍生物置于疏水性和位置时，特别能够显著增加这种折叠基序的稳定性。然而，尚未表明通过合理设计，氟化氨基酸是否可以用作控制 CC 组装过程的正交工具。在当前的工作中，我们通过创建基于先前在我们小组中建立和表征的 VPE/VPK 杂合 CC 系统的组合肽文库来解决这个问题。这个 CC 模型使我们能够筛选与 VPE/VPK 模型位置上的不同潜在结合伙伴相互作用的氟化氨基酸，特别强调研究α支链脂肪族氟化氨基酸侧链中的立体化学对 CC 性质（如寡聚状态、热力学稳定性和取向）的影响。利用圆二色性、尺寸排阻色谱法和Förster 共振能量转移测量法，对文库成员的 28 种组合进行了结构、寡聚和热稳定性的特征描述。这种详细的方法表明，基序的稳定性和寡聚状态不仅取决于相应氨基酸的立体需求和氟化，还取决于侧链中的立体化学。结果应用于氟化驱动的正交组装的合理设计，我们可以证明 CC 二聚体的形成是基于氟化氨基酸之间的特异性相互作用。这些结果表明，氟化氨基酸除了经典的静电和疏水相互作用外，还可以作为一种正交工具，用于微调和指导肽-肽相互作用。此外，在氟化氨基酸的空间中，我们可以证明不同氟化侧链之间相互作用的特异性。
