Base catalysis of chromophore formation in Arg96 and Glu222 variants of green fluorescent protein.
In green fluorescent protein (GFP), chromophore biosynthesis is initiated by a spontaneous main-chain condensation reaction. Nucleophilic addition of the Gly67 amide nitrogen to the Ser65 carbonyl carbon is catalyzed by the protein fold and leads to a heterocyclic intermediate. To investigate this mechanism, we substituted the highly conserved residues Arg96 and Glu222 in enhanced GFP (EGFP). In the R96M variant, the rate of chromophore formation is greatly reduced (time constant = 7.5 x 10(3) h, pH 7) and exhibits pH dependence. In the E222Q variant, the rate is also attenuated at physiological pH (32 h, pH 7) but is accelerated severalfold beyond that of EGFP at pH 9-10. In contrast, EGFP maturation is pH-independent and proceeds with a time constant of 1 h (pH 7-10). Mass spectrometric results for R96M and E222Q indicate accumulation of the pre-cyclization state, consistent with rate-limiting backbone condensation. The pH-rate profile implies that the Glu222 carboxylate titrates with an apparent pK(a) of 6.5 in R96M and that the Gly67 amide nitrogen titrates with an apparent pK(a) of 9.2 in E222Q. These data suggest a model for GFP chromophore synthesis in which the carboxylate of Glu222 plays the role of a general base, facilitating proton abstraction from the Gly67 amide nitrogen or the Tyr66 alpha-carbon. Arg96 fulfills the role of an electrophile by lowering the respective pK values and stabilizing the alpha-enolate. Modulating the base strength of the proton-abstracting group may aid in the design of fast-maturing GFPs with improved characteristics for real-time monitoring of cellular events.
Sniegowski
Jennifer A
JA
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA.
Lappe
Jason W
JW
Patel
Hetal N
HN
Huffman
Holly A
HA
Wachter
Rebekka M
RM
D000595:Amino Acid Sequence; D000838:Anions; D001120:Arginine / Q000737:chemistry*; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D018698:Glutamic Acid / Q000737:chemistry*; D005998:Glycine / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009584:Nitrogen / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011522:Protons; D012694:Serine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry; D014466:Ultraviolet Rays
D000838:Anions; D010455:Peptides; D011522:Protons; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D014443:Tyrosine; D012694:Serine; D002244:Carbon; D001120:Arginine; D014357:Trypsin; D009584:Nitrogen; D005998:Glycine