Evaluation of AlphaFold3 prediction for post-translational modification, oligomeric assembly, and quenchable metal binding of fluorescent proteins

Green fluorescent proteins (GFPs) are optical markers that are widely used in molecular and cell biology studies to track the location and function of biomolecules. Elucidating their structures will facilitate further engineering of these fluorescent proteins (FPs) to enhance their properties. AlphaFold3 (AF3) is a recently developed prediction tool that exhibits higher accuracy compared with other prediction tools, particularly in predicting protein–ligand interactions with state-of-the-art docking tools. However, studies on the use of AF3 to analyze the structure of FPs have not been fully conducted. To determine the accuracy of FP prediction using AF3, chromophore formation, oligomeric states, and metal ion binding of FPs were analyzed and compared with those of experimentally determined FPs. AF3 could not generate a chromophore comprising the two-ring structure by post-translational modification. Moreover, the oligomeric assembly formation of the FPs was similar to that of experimental oligomeric FPs; however, the detailed residue interactions between FP monomers were different. Quenchable metal ion docking to FPs using AF3 revealed a similar metal-binding site; however, metal coordination was significantly different between AF3 and the experimental structure. These results provide insight into the potential and limitations of using AF3 for FPs.