Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan; Medical Device Development and Regulation Research Center, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan; Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
Biochem Biophys Res Commun. 2021 Jul 23;563:54-59. doi: 10.1016/j.bbrc.2021.05.053. Epub 2021 May 28.
Proteins function through interactions with other molecules. In protein engineering, scientists often engineer proteins by mutating their amino acid sequences on the protein surface to improve various physicochemical properties. "Supercharging" is a method to design proteins by mutating surface residues with charged amino acids. Previous studies demonstrated that supercharging mutations conferred better thermal resistance, solubility, and cell penetration to proteins. Likewise, antibodies recognize antigens through the antigen-binding site on the surface. The genetic and structural diversity of antibodies leads to high specificity and affinity toward antigens, enabling antibodies to be versatile tools in various applications. When assessing therapeutic antibodies, surface charge is an important factor to consider because the isoelectric point plays a role in protein clearance inside the body. In this study, we explored how supercharging mutations affect physicochemical properties of antibodies. Starting from a crystal structure of an antibody with the net charge of -4, we computationally designed a supercharged variant possessing the net charge of +10. The positive-supercharged antibody exhibited marginal improvement in thermal stability, but the secondary structure and the binding affinity to the antigen (net charge of +8) were preserved. We also used physicochemical measurements and molecular dynamics simulations to analyze the effects of supercharging mutations in sodium phosphate buffer with different pH and ion concentrations, which revealed preferential solvation of phosphate ions to the supercharged surface relative to the wild-type surface. These results suggest that supercharging would be a useful approach to preserving thermal stability of antibodies in a wide range of pH, which may enable further diversification of antibody repertoires beyond natural evolution.
蛋白质通过与其他分子的相互作用发挥功能。在蛋白质工程中，科学家通常通过突变蛋白质表面的氨基酸序列来改善各种物理化学性质，从而对蛋白质进行工程改造。“超荷”是一种通过突变表面残基为带电荷的氨基酸来设计蛋白质的方法。先前的研究表明，超荷突变赋予蛋白质更好的热稳定性、溶解性和细胞穿透性。同样，抗体通过表面上的抗原结合位点识别抗原。抗体的遗传和结构多样性导致其对抗原具有高度的特异性和亲和力，使其成为各种应用中多功能的工具。在评估治疗性抗体时，表面电荷是一个需要考虑的重要因素，因为等电点在体内蛋白质清除中起作用。在这项研究中，我们探讨了超荷突变如何影响抗体的物理化学性质。我们从一个净电荷为-4 的抗体晶体结构开始，通过计算设计了一个净电荷为+10 的超荷变体。正超荷抗体在热稳定性方面略有改善，但二级结构和与抗原的结合亲和力（净电荷为+8）得以保留。我们还使用物理化学测量和分子动力学模拟来分析在不同 pH 值和离子浓度的磷酸钠缓冲液中，超荷突变的影响，结果表明相对于野生型表面，磷酸盐离子优先溶剂化超荷表面。这些结果表明，超荷可能是一种在广泛的 pH 值范围内保持抗体热稳定性的有用方法，这可能使抗体库的多样化进一步超越自然进化。
