A MOLECULAR MECHANICS AND DATABASE ANALYSIS OF THE STRUCTURAL PREORGANIZATION AND ACTIVATION OF THE CHROMOPHORE-CONTAINING HEXAPEPTIDE FRAGMENT IN GREEN FLUORESCENT PROTEIN

We propose that heterologous posttranslational chromophore formation i n green fluorescent protein (GFP) occurs because the chromophore-formi ng amino acid residues 65SYG67 are preorganized and activated for imid azolinone ring formation. Based on extensive molecular mechanical conf ormational searching of the precursor hexapeptide fragment (64FSYGVQ69 ), we suggest that the presence of low energy conformations characteri zed by shea contacts (similar to 3 Angstrom) between the carbonyl carb on of Ser65 and the amide nitrogen of Gly67 accounts for the initial s tep in posttranslational chromophore formation. Database searches show ed that the tight turn required to establish the key short contact is a unique structural motif that is rarely found, except in other FSYG t etrapeptide sequences. Additionally, ab initio calculations demonstrat ed that an arginine side chain can hydrogen bond to the carbonyl oxyge n of Ser65, activating this group for nucleophilic attack by the nearb y lone pair of the Gly67 amide nitrogen. We propose that GFP chromopho re-formation is initiated by a unique combination of conformational an d electronic enhancements, identified by computational methods.