González-Masís Jeimmy, Cubero-Sesin Jorge M, Guerrero Simón, González-Camacho Sara, Corrales-Ureña Yendry Regina, Redondo-Gómez Carlos, Vega-Baudrit José Roberto, Gonzalez-Paz Rodolfo J
Escuela de Ciencia e Ingeniería de los Materiales, Instituto Tecnológico de Costa Rica, Cartago, 159-7050, Costa Rica.
Instituto de Investigación Interdisciplinar en Ciencias Biomedicas SEK (I3CBSEK), Facultad de Ciencias de la Salud, Universidad SEK, Fernando Manterola 0789, 7500000, Santiago, Chile.
Biomater Res. 2020 Nov 23;24(1):19. doi: 10.1186/s40824-020-00197-0.
Collagen, the most abundant protein in the animal kingdom, represents a promising biomaterial for regenerative medicine applications due to its structural diversity and self-assembling complexity. Despite collagen's widely known structural and functional features, the thermodynamics behind its fibrillogenic self-assembling process is still to be fully understood. In this work we report on a series of spectroscopic, mechanical, morphological and thermodynamic characterizations of high purity type I collagen (with a D-pattern of 65 nm) extracted from Wistar Hannover rat tail. Our herein reported results can be of help to elucidate differences in self-assembly states of proteins using ITC to improve the design of energy responsive and dynamic materials for applications in tissue engineering and regenerative medicine.
Herein we report the systematic study on the self-assembling fibrillogenesis mechanism of type I collagen, we provide morphological and thermodynamic evidence associated to different self-assembly events using ITC titrations. We provide thorough characterization of the effect of pH, effect of salts and protein conformation on self-assembled collagen samples via several complementary biophysical techniques, including circular dichroism (CD), Fourier Transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), atomic force microscopy (AFM), scanning electron microscopy (SEM), dynamic mechanical thermal analysis (DMTA) and thermogravimetric analysis (TGA).
Emphasis was made on the use of isothermal titration calorimetry (ITC) for the thermodynamic monitoring of fibrillogenesis stages of the protein. An overall self-assembly enthalpy value of 3.27 ± 0.85 J/mol was found. Different stages of the self-assembly mechanism were identified, initial stages take place at pH values lower than the protein isoelectric point (pI), however, higher energy release events were recorded at collagen's pI. Denatured collagen employed as a control exhibited higher energy absorption at its pI, suggesting different energy exchange mechanisms as a consequence of different aggregation routes.
胶原蛋白是动物界中含量最丰富的蛋白质，由于其结构多样性和自组装复杂性，是再生医学应用中一种很有前景的生物材料。尽管胶原蛋白具有广为人知的结构和功能特征，但其纤维形成自组装过程背后的热力学仍有待充分理解。在这项工作中，我们报告了从Wistar Hannover大鼠尾巴中提取的高纯度I型胶原蛋白（具有65nm的D型图案）的一系列光谱、力学、形态学和热力学表征。我们在此报告的结果有助于阐明使用等温滴定量热法（ITC）来改善用于组织工程和再生医学的能量响应和动态材料设计时蛋白质自组装状态的差异。
在此我们报告了对I型胶原蛋白自组装纤维形成机制的系统研究，我们使用ITC滴定提供了与不同自组装事件相关的形态学和热力学证据。我们通过几种互补的生物物理技术，包括圆二色性（CD）、傅里叶变换红外光谱（FTIR）、差示扫描量热法（DSC）、原子力显微镜（AFM）、扫描电子显微镜（SEM）、动态机械热分析（DMTA）和热重分析（TGA），对pH值的影响、盐的影响和蛋白质构象对自组装胶原蛋白样品的影响进行了全面表征。
重点是使用等温滴定量热法（ITC）对蛋白质纤维形成阶段进行热力学监测。发现总体自组装焓值为3.27±0.85J/mol。确定了自组装机制的不同阶段，初始阶段发生在pH值低于蛋白质等电点（pI）时，然而，在胶原蛋白的pI处记录到更高的能量释放事件。用作对照的变性胶原蛋白在其pI处表现出更高的能量吸收，表明由于不同的聚集途径导致不同的能量交换机制。
