Mechanistic aspects of GFP chromophore biogenesis

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

We have investigated the autocatalytic mechanism of green fluorescent protein (GFP) maturation. To this end, we have used techniques such as site-directed mutagenesis, X-ray crystallography and in vitro kinetics, and have monitored the reaction by fluorescence, HPLC and MALDI (matrix-assisted laser desorption ionization) mass spectrometry. In summary, we find that chromophore formation, which generally occurs within 40 to 60 rain, can be accelerated dramatically under some conditions. In the E222Q variant, the rate-limiting process appears to be a function of slow proton transfer steps. Other mutagenesis data indicate that chromophore biogenesis is not driven by the aromatic character of residue 66. The GFP self-modification process involves a rate-limiting oxidation reaction that results in the production of H 2O2. The data are most consistent with a reaction mechanism that proceeds via cyclization-oxidation-dehydration during in vitro maturation under aerobic conditions. The ejection of water from the heterocycle that is formed from main chain protein atoms appears to depend on the degree of n-overlap of the five-membered ring with the side chain adduct.

Original languageEnglish (US)
Title of host publicationGenetically Engineered Probes for Biomedical Applications
DOIs
StatePublished - Jun 26 2006
EventGenetically Engineered Probes for Biomedical Applications - San Jose, CA, United States
Duration: Jan 24 2006Jan 24 2006

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume6098
ISSN (Print)1605-7422

Other

OtherGenetically Engineered Probes for Biomedical Applications
CountryUnited States
CitySan Jose, CA
Period1/24/061/24/06

Keywords

  • Chromophore biogenesis
  • Green fluorescent protein
  • Hydrogen peroxide evolution
  • Intrinsic cofactors
  • Tyrosine oxidation

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging

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  • Cite this

    Wachter, R. (2006). Mechanistic aspects of GFP chromophore biogenesis. In Genetically Engineered Probes for Biomedical Applications [609803] (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 6098). https://doi.org/10.1117/12.648542