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Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy.
Biochemistry. 1998 Dec 01; 37(48):16915-21.B

Abstract

Green Fluorescent Protein (GFP) is a bioluminescence protein from the jelly fish Aequorea victoria. It can exist in at least two spectroscopically distinct states: GFP395 and GFP480, with peak absorption at 395 and 480 nm, respectively, presumably resulting from a change in the protonation state of the phenolic ring of its chromophore. When GFP is formed upon heterologous expression in Escherichia coli, its chromophore is mainly present as the neutral species. UV and visible light convert (the chromophore of) GFP quantitatively from this neutral- into the anionic form. On the basis of X-ray diffraction, it was recently proposed (Brejc, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2306-2311; Palm, G. J. et al. (1997) Nat. Struct. Biol. 4, 361-365) that the carboxylic group of Glu222 functions as the proton acceptor of the chromophore of GFP, during the transition from the neutral form (i.e., GFP395) to the ionized form (GFP480). However, X-ray crystallography cannot detect protons directly. The results of FTIR difference spectroscopy, in contrast, are highly sensitive to changes in the protonation state between two conformations of a protein. Here we report the first characterization of GFP, and its photoconversion, with FTIR spectroscopy. Our results clearly show the change in protonation state of the chromophore upon photoconversion. However, they do not provide indications for a change of the protonation state of a glutamate side chain between the states GFP395 and GFP480, nor for an isomerization of the double bond that forms part of the link between the two rings of the chromophore.

Authors+Show Affiliations

Laboratories for Microbiology and for Biochemistry, E.C. Slater Institute, University of Amsterdam, Amsterdam, The Netherlands.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9836584

Citation

van Thor, J J., et al. "Characterization of the Photoconversion of Green Fluorescent Protein With FTIR Spectroscopy." Biochemistry, vol. 37, no. 48, 1998, pp. 16915-21.
van Thor JJ, Pierik AJ, Nugteren-Roodzant I, et al. Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy. Biochemistry. 1998;37(48):16915-21.
van Thor, J. J., Pierik, A. J., Nugteren-Roodzant, I., Xie, A., & Hellingwerf, K. J. (1998). Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy. Biochemistry, 37(48), 16915-21.
van Thor JJ, et al. Characterization of the Photoconversion of Green Fluorescent Protein With FTIR Spectroscopy. Biochemistry. 1998 Dec 1;37(48):16915-21. PubMed PMID: 9836584.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy. AU - van Thor,J J, AU - Pierik,A J, AU - Nugteren-Roodzant,I, AU - Xie,A, AU - Hellingwerf,K J, PY - 1998/12/4/pubmed PY - 1998/12/4/medline PY - 1998/12/4/entrez SP - 16915 EP - 21 JF - Biochemistry JO - Biochemistry VL - 37 IS - 48 N2 - Green Fluorescent Protein (GFP) is a bioluminescence protein from the jelly fish Aequorea victoria. It can exist in at least two spectroscopically distinct states: GFP395 and GFP480, with peak absorption at 395 and 480 nm, respectively, presumably resulting from a change in the protonation state of the phenolic ring of its chromophore. When GFP is formed upon heterologous expression in Escherichia coli, its chromophore is mainly present as the neutral species. UV and visible light convert (the chromophore of) GFP quantitatively from this neutral- into the anionic form. On the basis of X-ray diffraction, it was recently proposed (Brejc, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2306-2311; Palm, G. J. et al. (1997) Nat. Struct. Biol. 4, 361-365) that the carboxylic group of Glu222 functions as the proton acceptor of the chromophore of GFP, during the transition from the neutral form (i.e., GFP395) to the ionized form (GFP480). However, X-ray crystallography cannot detect protons directly. The results of FTIR difference spectroscopy, in contrast, are highly sensitive to changes in the protonation state between two conformations of a protein. Here we report the first characterization of GFP, and its photoconversion, with FTIR spectroscopy. Our results clearly show the change in protonation state of the chromophore upon photoconversion. However, they do not provide indications for a change of the protonation state of a glutamate side chain between the states GFP395 and GFP480, nor for an isomerization of the double bond that forms part of the link between the two rings of the chromophore. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9836584/Characterization_of_the_photoconversion_of_green_fluorescent_protein_with_FTIR_spectroscopy_ L2 - https://doi.org/10.1021/bi981170f DB - PRIME DP - Unbound Medicine ER -