Li Baoshan, Song Junyi, Mao Ting, Zeng Ling, Ye Zonghuang, Hu Biru
College of Science, National University of Defense Technology, Changsha, China.
Logistics Center, National University of Defense Technology, Changsha, China.
Front Bioeng Biotechnol. 2022 Oct 12;10:998194. doi: 10.3389/fbioe.2022.998194. eCollection 2022.
Barnacles are typical fouling organisms strongly adhere to immersed solid substrates by secreting proteinaceous adhesives called cement proteins (CPs). The self-assembly of the CPs forms a permanently bonded layer that binds barnacles to foreign surfaces. However, it is difficult to determine their natural structure and describe their self-assembly properties due to the abundance of cysteines in whole-length CP20. A putative functional motif of CP20 (BalCP20) was identified to present distinctive self-assembly and wet-binding characteristics. Atomic-force microscopy (AFM) and transmission electron microscope (TEM) investigations showed that wildtype BalCP20-P3 formed grain-like spindles, which assembled into fractal-like structures like ears of wheat. SDS-PAGE, AFM, and LSCM showed that DTT treatment opened up disulfide bonds between cysteines and disrupted fractal-like structures. Additionally, these morphologies were abolished when one of the BalCP20-P3 four cysteines was mutated by alanine. Circular dichroism (CD) results suggested that the morphological diversity among BalCP20-P3 and its mutations was related to the proportion of -helices. Finally, quartz crystal microbalance with dissipation (QCM-D) detected that BalCP20-P3 and its mutations with diverse self-assemblies occupied different affinities. The above results demonstrated that cysteines and disulfide bonds played a crucial role in the self-assembly and wet binding of BalCP20-P3. The work provides new ideas for the underwater bonding of BalCP20 and developing new bionic underwater adhesives.
藤壶是典型的污损生物，通过分泌称为水泥蛋白（CPs）的蛋白质类粘合剂，牢固地附着在浸入水中的固体基质上。CPs的自组装形成了一个永久结合层，将藤壶与外来表面结合在一起。然而，由于全长CP20中富含半胱氨酸，很难确定其天然结构并描述其自组装特性。已鉴定出CP20的一个假定功能基序（BalCP20）具有独特的自组装和湿结合特性。原子力显微镜（AFM）和透射电子显微镜（TEM）研究表明，野生型BalCP20-P3形成颗粒状纺锤体，这些纺锤体组装成类似麦穗的分形结构。SDS-PAGE、AFM和激光扫描共聚焦显微镜（LSCM）表明，二硫苏糖醇（DTT）处理打开了半胱氨酸之间的二硫键，破坏了分形结构。此外，当BalCP20-P3的四个半胱氨酸之一被丙氨酸突变时，这些形态消失。圆二色性（CD）结果表明，BalCP20-P3及其突变体之间的形态多样性与α-螺旋的比例有关。最后，石英晶体微天平与耗散监测技术（QCM-D）检测到，具有不同自组装的BalCP-20-P3及其突变体具有不同的亲和力。上述结果表明，半胱氨酸和二硫键在BalCP20-P3的自组装和湿结合中起关键作用。这项工作为BalCP20的水下粘结和开发新型仿生水下粘合剂提供了新思路。
