Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein

Rebekka M. Wachter, Brett A. King, Roger Heim, Karen Kallio, Roger Y. Tsien, Steven G. Boxer, S. James Remington

Research output: Contribution to journalArticle

142 Scopus citations

Abstract

The crystal structure of a blue emission variant (Y66H/Y145F) of the Aequorea victoria green fluorescent protein has been determined by molecular replacement and the model refined. The crystallographic R-factor is 18.1% for all data from 20 to 2.1 Å, and the model geometry is-excellent. The chromophore is non-native and is autocatalytically generated from the internal tripeptide Ser65-His66-Gly67. The final electron density maps indicate that the formation of the chromophore is complete, including 1,2 dehydration of His66 as indicated by the planarity of the chromophore. The chromophore is in the cis conformation, with no evidence for any substantial fraction of the trans configuration or uncyclized apoprotein, and is well- shielded from bulk solvent by the folded protein. These characteristics indicate that the machinery for production of the chromophore from a buried tripeptide unit is not only intact but also highly efficient in spite of a major change in chromophore chemical structure. Nevertheless, there are significant rearrangements in the hydrogen bond configuration around the chromophore as compared to wild-type, indicating flexibility of the active site. pH titration of the intact protein and the chromopeptide (pK(a1) = 4.9 ± 0.1, pK(a2) = 12.0 ± 0.1) suggests that the predominant form of the chromophore in the intact protein is electrically neutral. In contrast to the wild-type protein [Chattoraj, M., King, B. A., Bublitz, G. U., and Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A., 8362-8367]; femtosecond fluorescence up-conversion spectroscopy of the intact protein and a partially deuterated form strongly suggests that excited-state proton transfer is not coupled to fluorescence emission.

Original languageEnglish (US)
Pages (from-to)9759-9765
Number of pages7
JournalBiochemistry
Volume36
Issue number32
DOIs
StatePublished - Aug 12 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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    Wachter, R. M., King, B. A., Heim, R., Kallio, K., Tsien, R. Y., Boxer, S. G., & Remington, S. J. (1997). Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein. Biochemistry, 36(32), 9759-9765. https://doi.org/10.1021/bi970563w