Influence of nanobody binding on fluorescence emission, mobility, and organization of GFP-tagged proteins

Abstract

Summary Advanced fluorescence microscopy studies require specific and monovalent molecular labeling with bright and photostable fluorophores. This necessity led to the widespread use of fluorescently labeled nanobodies against commonly employed fluorescent proteins (FPs). However, very little is known how these nanobodies influence their target molecules. Here, we tested commercially available nanobodies and observed clear changes of the fluorescence properties, mobility and organization of green fluorescent protein (GFP) tagged proteins after labeling with the anti-GFP nanobody. Intriguingly, we did not observe any co-diffusion of fluorescently labeled nanobodies with the GFP-labeled proteins. Our results suggest significant binding of the nanobodies to a non-emissive, likely oligomerized, form of the FPs, promoting disassembly into monomeric form after binding. Our findings have significant implications on the application of nanobodies and GFP labeling for studying dynamic and quantitative protein organization in the plasma membrane of living cells using advanced imaging techniques.

Light-guided intrabodies for on-demand in situ target recognition in human cells

“…Furthermore, it remains unclear whether the uncontrolled binding of nanobodies interferes with the function, assembly, and subcellular dynamics of intracellular target proteins before the actual analysis. 16 To surpass possible artifacts derived from premature binding, a major advancement would be to control intrabody binding by light in living mammalian cells.…”

The transmembrane domain of the amyloid precursor protein is required for antiamyloidogenic processing by α-secretase ADAM10

“…S3 ). GFP-self-quenching is probably responsible for this effect, which can occur upon oligomerization of GFP-labeled proteins ( 39 , 40 ).…”

The mesoscale organization of syntaxin 1A and SNAP25 is determined by SNARE–SNARE interactions

“…In a previous study, the antibody accessibility of SNAP25 was strongly dependent on the conformational state of the protein; antibody staining was reduced by ~90 % after Ca 2+ -induced protein clustering. Moreover, SNAP25 clustering reduced the emission of its attached fluorescent protein, most likely due to self-quenching ( Zilly et al, 2011 ), an effect described to occur in GFP-labelled oligomers ( Ochiishi et al, 2016 ; Schneider et al, 2021 ). In the following, we test whether elevation of syntaxin 1A has any influence on the conformational state of SNAP25, possibly reducing its antibody accessibility.…”

“…However, apart from the possibility that GFP may affect molecule packing, self-quenching of close (e.g. less than 5 nm) fluorophores results in signal underestimation ( Ochiishi et al, 2016 ; Schneider et al, 2021 ). Consequently, not all molecules in a tightly packed crowd are detected.…”

“…Moreover, SNAP25 clustering reduced the emission of its attached fluorescent protein, most likely due to self-quenching (Zilly et al, 2011), an effect described to occur in GFP-labeled oligomers (Ochiishi et al, 2016;Schneider et al, 2021). In the following, we test whether elevation of syntaxin 1A has any influence on the conformational state of SNAP25, possibly reducing its antibody accessibility.…”