Constant-pH Molecular Dynamics Rationalizes Photoinduced Decarboxylation in Fluorescent Proteins

Many advanced imaging techniques, such as super-resolution microscopy, rely on photoinduced transformations of fluorescent proteins. In photoinduced decarboxylation, light absorption triggers loss of CO 2 from an acidic residue near the chromophore, often accompanied by a change in chromophore protonation that switches the protein to a fluorescent form, as in PA-GFP. Evidence from proteins in which this reaction has been observed indicates that the decarboxylating residue must be present in its deprotonated form. Because protonation states are difficult to determine experimentally, here we perform constant-pH molecular dynamics (CpHMD) simulations on a set of fluorescent protein variants, and find that the predicted protonation states are consistent with the observed photoinduced properties. This includes superfolder GFP, which does not decarboxylate, and its T203D mutant, in which two residues undergo decarboxylation. CpHMD therefore offers a practical route to predicting which variants are capable of photoinduced decarboxylation and to designing new photoactivatable proteins.