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Nb2 exhibited the highest melting temperature (Tm); thus, Nb2 is a promising GFP nanobody candidate for use in applications requiring harsh testing conditions. We also compared the binding sites of available GFP nanobodies and showed that some of them can simultaneously bind with GFP and assemble into multifunctional complexes to manipulate GFP-tagged target proteins. Our results provide atomic-scale binding information for Nb2-sfGFP, which is important for the further development of GFP-nanobody based fusion protein manipulation techni
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