Center for Interdisciplinary Biosciences, Technology and Innovation Park, P.J. Šafárik University, Košice, Slovakia.
Department of Biophysics, Faculty of Science, P.J. Šafárik University, Košice, Slovakia.
Protein Sci. 2023 Aug;32(8):e4722. doi: 10.1002/pro.4722.
Recombinant spider silk proteins can be prepared in scalable fermentation processes and have been proven as sources of biomaterials for biomedical and technical applications. Nanofibrils, formed through the self-assembly of these proteins, possess unique structural and mechanical properties, serving as fundamental building blocks for the fabrication of micro- and nanostructured scaffolds. Despite significant progress in utilizing nanofibrils-based morphologies of recombinant spider silk proteins, a comprehensive understanding of the molecular mechanisms of nanofibrils self-assembly remains a challenge. Here, a detailed kinetic study of nanofibril formation from a recombinant spider silk protein eADF4(C16) in dependence on the protein concentration, seeding, and temperature is provided. For the global fitting of kinetic data obtained during the fibril formation, we utilized the online platform AmyloFit. Evaluation of the data revealed that the self-assembly mechanism of recombinant spider silk is dominated by secondary nucleation. Thermodynamic analyses show that both primary and secondary nucleations, as well as the elongation step of the eADF4(C16), are endothermic processes.
重组蜘蛛丝蛋白可以通过可扩展的发酵工艺制备，并已被证明是生物医学和技术应用中生物材料的来源。这些蛋白质通过自组装形成纳米原纤维，具有独特的结构和机械性能，可用作制造微纳米结构支架的基本构建块。尽管在利用基于重组蜘蛛丝蛋白纳米原纤维的形态方面已经取得了重大进展，但对纳米原纤维自组装的分子机制仍存在挑战。在这里，我们提供了详细的动力学研究，研究了重组蜘蛛丝蛋白 eADF4(C16) 在依赖于蛋白质浓度、种子和温度的情况下形成纳米原纤维的过程。为了对纤维形成过程中获得的动力学数据进行全局拟合，我们利用了在线平台 AmyloFit。对数据的评估表明，重组蜘蛛丝的自组装机制主要由二级成核主导。热力学分析表明，一级和二级成核以及 eADF4(C16) 的延伸步骤都是吸热过程。
