The detection of changes in the fluorescence characteristics of eGFP upon interaction with DARPins and Nanobodies has been scrutinized. Detected fluctuations in eGFP fluorescence due to interactions with engineered protein binders were analyzed, as presented in our multi-device data anlysis section. Aiming to comprehend the underlying mechanism and suspecting the involvement of subtle structural modifications, we extracted structures involving antibody-like proteins bound to green fluorescent protein (GFP) variants. A selection thereof is presented below. The analysis of binding sites, as demonstrated below, was seamlessly integrated into our sequence analysis scripts, as detailed in the corresponding section on sequence analysis.
Subsequently, we captured the interaction of amino acids in closest proximity to the chromophore. We automatically measured distances, priming them for subsequent utilization in R scripts. This becomes particularly relevant since certain structures indicate the presence of multiple potential arrangements, which aligns logically with the diverse states exhibited by eGFP. One example is found below in the bottom left corner.
While visually appealing, the depiction of interactions in three dimensions, as illustrated above, can be challenging to comprehend. To enhance clarity, we have condensed this information into bar graphs, enabling a quick identification of specific patterns of changes. Through this approach, we have discerned various distinct patterns and successfully determined the significance of certain residues in modulating the ratio between different GFP states.
