Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan.
Int J Biol Macromol. 2016 Oct;91:151-7. doi: 10.1016/j.ijbiomac.2016.05.074. Epub 2016 May 21.
Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.
蛋白质结构动力学对于理解结构与功能的关系至关重要。抗体可以识别其抗原，并通过亲和力成熟过程进化为免疫原以增加结合强度。在这项研究中，设计了一种针对（4-羟基-3-硝基苯乙酰基）的单链 Fv（scFv）抗体，该抗体源自亲和力成熟的类型 C6，由轻链和重链的可变区通过（GGGGS）3 连接肽连接而成。该 scFv 在大肠杆菌中以不溶性部分表达，在存在脲的情况下溶解，并通过逐步透析复性。正确复性的 scFv 被纯化，并使用分析超速离心、圆二色性光谱法、差示扫描量热法和表面等离子体共振生物传感器分析其结构、物理和功能特性。由于有利的焓贡献，抗原结合大大增加了 C6 scFv 的热稳定性。抗原结合动力学与完整的 C6 抗体相当。使用衍射 X 射线跟踪法分析结构动力学，表明抗原结合时波动受到抑制。从角扩散系数确定的抗原结合能与从动力学分析计算的抗原结合能吻合良好，表明在单分子水平检测到的波动很好地反映了抗原结合事件。
