More Haresh T, Zhang Kevin S, Srivastava Nikita, Frezzo Joseph A, Montclare Jin K
†Department of Chemical and Biomolecular Engineering, New York University Polytechnic School of Engineering, Brooklyn, New York 11201, United States.
∫Department of Biochemistry, SUNY Downstate Medical Center, Brooklyn, New York 11203, United States.
Biomacromolecules. 2015 Apr 13;16(4):1210-7. doi: 10.1021/bm5019062. Epub 2015 Apr 2.
We describe the design and characterization of fluorinated coiled-coil proteins able to assemble into robust nano- and microfibers. Fluorination is achieved biosynthetically by residue-specific incorporation of 5,5,5-trifluoroleucine (TFL). The fluorinated proteins C+TFL and Q+TFL are highly α-helical as confirmed via circular dichroism (CD) and more resistant to thermal denaturation compared to their nonfluorinated counterparts, C and Q. The fluorinated proteins demonstrate enhanced fiber assembly at pH 8.0 with higher order structure in contrast to nonfluorinated proteins, which are unable to form fibers under the same conditions. Ionic strength dependent fiber assembly is observed for fluorinated as well as wild-type proteins in which the fluorinated proteins exhibited more stable, thicker fibers. The fluorinated and nonfluorinated proteins reveal metal ion-dependent small molecule recognition and supramolecular assemblies. In the presence of Zn (II), enhanced thermal stability and fiber assembly is observed for the fluorinated proteins and their nonfluorinated counterparts. Whereas Ni (II) promotes aggregation with no fiber assembly, the stabilization of α-helix by Zn (II) results in enhanced binding to curcumin by the fluorinated proteins. Surprisingly, the nonfluorinated proteins exhibit multiple-fold increase in curcumin binding in the presence of Zn (II). In the context of the growing number of protein-based fiber assemblies, these fluorinated coiled-coil proteins introduce a new paradigm in the development of highly stable, robust self-assembling fibers under more physiologically relevant pH conditions that promotes the binding and release of small molecules in response to external cues.
我们描述了能够组装成坚固的纳米和微纤维的氟化卷曲螺旋蛋白的设计与特性。通过5,5,5-三氟亮氨酸（TFL）的残基特异性掺入实现生物合成氟化。通过圆二色性（CD）证实，氟化蛋白C+TFL和Q+TFL具有高度的α-螺旋结构，并且与未氟化的对应物C和Q相比，对热变性更具抗性。与未氟化的蛋白不同，氟化蛋白在pH 8.0时表现出增强的纤维组装和更高阶的结构，未氟化的蛋白在相同条件下无法形成纤维。观察到氟化蛋白和野生型蛋白都存在离子强度依赖性纤维组装，其中氟化蛋白表现出更稳定、更粗的纤维。氟化和未氟化的蛋白都揭示了金属离子依赖性的小分子识别和超分子组装。在存在Zn（II）的情况下，观察到氟化蛋白及其未氟化的对应物的热稳定性和纤维组装增强。而Ni（II）促进聚集但不形成纤维组装，Zn（II）对α-螺旋的稳定作用导致氟化蛋白与姜黄素的结合增强。令人惊讶的是，未氟化的蛋白在存在Zn（II）的情况下与姜黄素的结合增加了数倍。在基于蛋白质的纤维组装数量不断增加的背景下，这些氟化卷曲螺旋蛋白在更生理相关的pH条件下开发高度稳定、坚固的自组装纤维方面引入了一种新的范例，这种条件促进了小分子响应外部线索的结合和释放。
