期刊
NANO LETTERS
卷 22, 期 1, 页码 179-187出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.nanolett.1c03584
关键词
atomic force microscopy; protein engineering; single-molecule force spectroscopy; mechanical anisotropy; click chemistry; Go-Martini model; PCA
类别
资金
- University of Basel
- ETH Zurich
- ERC Starting Grant [MMA-715207]
- NCCR in Molecular Systems Engineering
- Swiss National Science Foundation [200021_175478]
- National Science Centre, Poland [2017/26/D/NZ1/00466]
- Foundation for Polish Science [MAB PLUS/11/2019]
- Swiss National Science Foundation (SNF) [200021_175478] Funding Source: Swiss National Science Foundation (SNF)
Using single-molecule AFM force spectroscopy in combination with click chemistry, the mechanical interactions between anticalin and its target CTLA-4 were studied. Results showed that pulling from different anchor residues significantly affected rupture forces and dissociation rates. Molecular dynamics simulations revealed a geometric dependency of mechanostability.
We used single-molecule AFM force spectroscopy (AFM-SMFS) in combination with click chemistry to mechanically dissociate anticalin, a non-antibody protein binding scaffold, from its target (CTLA-4), by pulling from eight different anchor residues. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in k(off) by 2-3 orders of magnitude over a force range of 50-200 pN. Five of the six internal anchor points gave rise to complexes significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1-10 nN s(-1). Anisotropic network modeling and molecular dynamics simulations helped to explain the geometric dependency of mechanostability. These results demonstrate that optimization of attachment residue position on therapeutic binding scaffolds can provide large improvements in binding strength, allowing for mechanical affinity maturation under shear stress without mutation of binding interface residues.
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