School of Chemistry, University of East Anglia, Norwich, UK.
Proteomics Facility, The John Innes Centre, Norwich, UK.
Nat Chem Biol. 2023 Jun;19(6):740-749. doi: 10.1038/s41589-022-01251-9. Epub 2023 Feb 16.
Design of biomolecules that perform two or more distinct functions in response to light remains challenging. Here, we have introduced concurrent photoactivity and photoreactivity into an epidermal growth factor receptor (EGFR)-targeting antibody fragment, 7D12. This was achieved by site-specific incorporation of photocaged tyrosine (pcY) for photoactivity and p-benzoyl-ʟ-phenylalanine (Bpa) for photoreactivity into 7D12. We identified a position for installing Bpa in 7D12 that has minimal effect on 7D12-EGFR binding affinity in the absence of light. Upon exposure to 365-nm light, this Bpa-containing 7D12 mutant forms a covalent bond with EGFR in an antigen-specific manner. We then developed a method for site-specific incorporation of pcY and Bpa at two distinct sites in 7D12. Finally, we demonstrated that in the absence of light, this pcY- and Bpa-containing mutant of 7D12 does not bind to EGFR, but irradiation with 365-nm light activates (1) specific binding and (2) covalent bond formation with EGFR.
设计能够对光做出两种或更多种不同响应的生物分子仍然具有挑战性。在这里，我们将光活性和光反应性同时引入表皮生长因子受体 (EGFR) 靶向抗体片段 7D12 中。这是通过将光封闭酪氨酸 (pcY) 和对苯甲酰基-L-苯丙氨酸 (Bpa) 定点掺入到 7D12 中实现的。我们确定了一个在不影响光下 7D12-EGFR 结合亲和力的情况下安装 Bpa 的位置。暴露于 365nm 光下后，这种含有 Bpa 的 7D12 突变体以抗原特异性的方式与 EGFR 形成共价键。然后，我们开发了一种在 7D12 中的两个不同位点定点掺入 pcY 和 Bpa 的方法。最后，我们证明在没有光的情况下，7D12 中的这种含有 pcY 和 Bpa 的突变体不会与 EGFR 结合，但用 365nm 光照射会激活 (1) 与 EGFR 的特异性结合和 (2) 与 EGFR 的共价键形成。
