Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants.

ABSTRACT: The maturation process of green fluorescent protein (GFP) entails a protein oxidation reaction triggered by spontaneous backbone condensation. The chromophore is generated by full conjugation of the Tyr66 phenolic group with the heterocycle, a process that requires C-H bond scission at the benzylic carbon. We have prepared isotope-enriched protein bearing tyrosine residues deuterated at the beta carbon, and have determined kinetic isotope effects (KIEs) on the GFP self-processing reaction. Progress curves for the production of H 2O 2 and the mature chromophore were analyzed by global curve fitting to a three-step mechanism describing preoxidation, oxidation and postoxidation events. Although a KIE for protein oxidation could not be discerned ( k H/ k D = 1.1 +/- 0.2), a full primary KIE of

SUBMITTER: Pouwels LJ 

PROVIDER: S-EPMC2643082 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature