Extreme thermal stability of the antiGFP nanobody - GFP complex

The green fluorescent protein (GFP) and its derivatives are widely used in biomedical research. The manipulation of GFP-tagged proteins by GFP-specific binders, e.g. single-domain antibodies (nanobodies), is of increasing significance. It is therefore important to better understand the properties of antiGFP-GFP interaction in order to establish methodological applications. In this work the interaction of superfolder GFP (sfGFP) and its enhancer nanobody (aGFP_enh) was characterized further.

Previous calorimetric experiments demonstrated that the aGFP_enh nanobody binds strongly to sfGFP with a nanomolar affinity. Here we show that this interaction results in a substantial structural stabilization of aGFP_enh reflected in a significant increase of its melting temperature by almost 30 °C. The thermal stability of the sfGFP-aGFP_enh complex is close to 85 °C in the pH range 7.0-8.5. For therapeutic applications thermoresistance is often an essential factor. Our results suggest that methodologies based on GFP-aGFP interaction can be applied under a wide range of physicochemical conditions. The aGFP_enh nanobody seems to be suitable for manipulating sfGFP-labeled targets even in extreme thermophilic organisms.