Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How “Molecular Surgery” of the Backbone Affects Folding and Stability

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.