Structural insights into two distinct nanobodies recognizing the same epitope of green fluorescent protein.

Biochemical and Biophysical Research Communications
Peiyu ZhongYu Ding

Abstract

Green fluorescent protein (GFP) and its derivatives are widely used in biomedical research, and the manipulation of GFP-tagged proteins by GFP-specific binders is highly desired. However, structural information on how these binders bind with GFP is still lacking. In this study, we determined the crystal structure of the nanobody Nb2 complexed with superfolder GFP (sfGFP) at a resolution of 2.2 Å. Interestingly, although the complementarity-determining regions (CDRs) of Nb2 and LaG16 sequences were only 29.7% identical, they both bound to the same epitope of GFP and existed in the same orientation. Structural analysis indicated that they achieved similar binding characteristics through different mechanisms. We further verified the kinetics and thermodynamics of binding by biolayer interferometry (BLI) and isothermal titration calorimetry (ITC). Nb2 showed a slightly higher binding affinity for sfGFP than LaG16. The stability of GFP-specific nanobodies was verified by nano differential scanning fluorimetry (nanoDSF). 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 nano...Continue Reading

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Citations

Sep 26, 2021·Protein Science : a Publication of the Protein Society·Ziying WangYu Ding

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