Yue, Z.; Li, Y.; Cai, H.; Yao, H.; Li, D.; Ni, A.; Li, T.

Green fluorescence protein (GFP) lights up almost every aspects of life sciences. An ultra thermostable GFP (TGP), engineered from a coral GFP, offers potential advantages over traditional jellyfish-derived GFP due to its high stability. However, owing to its later discovery, TGP lacks the extensive toolsets available for GFP, such as heavy chain-only antibody binders known as nanobodies. In this study, we have structurally characterized TGP in complex with Sb92, a synthetic nanobody identified from a previous in vitro screening, revealing its precise three-dimensional epitope. This structural insight, alongside the previously characterized Sb44-TGP complex, have allowed us to rationally design disulfide bonds between antigen and antibody for tighter interactions. Using biochemical analyses, we identify two bridged complexes (TGP A18C-Sb44 V100C, TGP E118C-Sb92 S57C), with the TGP-Sb92 disulfide pair showing high resistance to reducing agents. Our study expands the toolkit available for TGP and should encourage its wider applications.