Shin Donghyuk, Kim Gwanho, Kim Gyuhee, Zheng Xu, Kim Yang-Gyun, Lee Sangho
Department of Biological Sciences, Sungkyunwan University, Suwon, Republic of Korea.
Department of Chemistry, Sungkyunwan University, Suwon, Republic of Korea.
Biochem Biophys Res Commun. 2014 Dec 12;455(3-4):339-46. doi: 10.1016/j.bbrc.2014.11.013. Epub 2014 Nov 15.
Coiled coil has served as an excellent model system for studying protein folding and developing protein-based biomaterials. Most designed coiled coils function as oligomers, namely intermolecular coiled coils. However, less is known about structural and biochemical behavior of intramolecular coiled coils where coiled coil domains are covalently linked in one polypeptide. Here we prepare a protein which harbors three coiled coil domains with two short linkers, termed intramolecular tandem coiled coil (ITCC) and characterize its structural and biochemical behavior in solution. ITCC consists of three coiled coil domains whose sequences are derived from Coil-Ser and its domain swapped dimer. Modifications include positioning E (Glu) residue at "e" and K (Lys) at "g" positions throughout heptad repeats to enhance ionic interaction among its constituent coiled coil domains. Molecular modeling of ITCC suggests a compact triple helical bundle structure with the second and the third coiled coil domains forming a canonical coiled coil. ITCC exists as a mixture of monomeric and dimeric species in solution. Small-angle X-ray scattering reveals ellipsoidal molecular envelopes for both dimeric and monomeric ITCC in solution. The theoretically modeled structures of ITCC dock well into the envelopes of both species. Higher ionic strength shifts the equilibrium into monomer with apparently more compact structure while secondary structure remains unchanged. Taken together, our results suggest that our designed ITCC is predominantly monomeric structure through the enhanced ionic interactions, and its conformation is affected by the concentration of ionic species in the buffer.
卷曲螺旋一直是研究蛋白质折叠和开发基于蛋白质的生物材料的优秀模型系统。大多数设计的卷曲螺旋作为寡聚体发挥作用，即分子间卷曲螺旋。然而，对于分子内卷曲螺旋的结构和生化行为了解较少，在分子内卷曲螺旋中，卷曲螺旋结构域在一条多肽链中通过共价键相连。在这里，我们制备了一种含有三个卷曲螺旋结构域和两个短连接子的蛋白质，称为分子内串联卷曲螺旋（ITCC），并对其在溶液中的结构和生化行为进行了表征。ITCC由三个卷曲螺旋结构域组成，其序列源自Coil-Ser及其结构域交换二聚体。修饰包括在整个七肽重复序列的“e”位定位E（Glu）残基，在“g”位定位K（Lys）残基，以增强其组成卷曲螺旋结构域之间的离子相互作用。ITCC的分子模型表明其具有紧凑的三螺旋束结构，其中第二个和第三个卷曲螺旋结构域形成典型的卷曲螺旋。ITCC在溶液中以单体和二聚体形式混合存在。小角X射线散射揭示了溶液中ITCC二聚体和单体的椭圆形分子包络。ITCC的理论建模结构很好地对接入两种形式的包络中。更高的离子强度将平衡转移到具有明显更紧凑结构的单体中，而二级结构保持不变。综上所述，我们的结果表明，我们设计的ITCC通过增强的离子相互作用主要为单体结构，其构象受缓冲液中离子种类浓度的影响。
