Department of Applied Science, University of California, Davis, CA 95616-8816, USA.
Protein Eng Des Sel. 2011 Sep;24(9):649-57. doi: 10.1093/protein/gzr029. Epub 2011 Jul 5.
Antibody single-chain variable fragments (scFvs) offer particular advantages over the full-size antibodies, including easy expression, efficient local concentration and fast body clearance. However, scFvs typically show low thermal stability that limits their biomedical and biotechnological applications. In this study, we examined the thermal stability of the human and murine vascular endothelial growth factor antibody scFv fragment by molecular dynamics simulations. A consistent observation was the dissociation of the light-chain (VL) and heavy-chain (VH) domains and loss of the native structures of both domains in the simulations at the elevated temperatures. The stability-limiting structural elements in the protein were revealed from the detailed analyses on the native contacts. We found that dissociation of the VL-VH domains was the first event leading to the unfolding of the native structure of the protein and the disruption of the VL-VH interface was largely due to the break of the interfacial hydrophobic and aromatic interactions while the hydrogen-bonding interaction between Gln38 in VL and Gln39 in VH remained. Within the β-barrel structure of the VL and VH domains, β-strands β6, β2 and β11 appeared to be the least stable. In addition, we found that the VH domain was more thermally resistant than the VL domain. Based on these findings, we discussed potential strategies to improve the stability of this therapeutically important scFv fragment.
抗体单链可变片段 (scFv) 相对于完整抗体具有特殊优势，包括易于表达、高效局部浓度和快速清除。然而，scFv 通常表现出较低的热稳定性，限制了它们在生物医学和生物技术中的应用。在这项研究中，我们通过分子动力学模拟研究了人源和鼠源血管内皮生长因子抗体 scFv 片段的热稳定性。一致的观察结果是，在模拟升温过程中，轻链 (VL) 和重链 (VH) 结构域解离，两个结构域的天然结构丧失。通过对天然接触的详细分析揭示了蛋白质中稳定性限制的结构元件。我们发现，VL-VH 结构域的解离是导致蛋白质天然结构展开的第一个事件，VL-VH 界面的破坏主要是由于界面疏水性和芳香性相互作用的破坏，而 VL 中的 Gln38 和 VH 中的 Gln39 之间的氢键相互作用仍然存在。在 VL 和 VH 结构域的β-桶结构中，β6、β2 和 β11 似乎是最不稳定的。此外，我们发现 VH 结构域比 VL 结构域具有更高的耐热性。基于这些发现，我们讨论了提高这种治疗重要 scFv 片段稳定性的潜在策略。
