Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy.
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.
van Thor
J J
JJ
Laboratories for Microbiology and for Biochemistry, E.C. Slater Institute, University of Amsterdam, Amsterdam, The Netherlands.
Pierik
A J
AJ
Nugteren-Roodzant
I
I
Xie
A
A
Hellingwerf
K J
KJ
D001597:Benzylidene Compounds / Q000528:radiation effects*; D049452:Green Fluorescent Proteins; D008027:Light; D008164:Luminescent Proteins / Q000235:genetics / Q000528:radiation effects*; D010860:Pigments, Biological / Q000528:radiation effects*; D011522:Protons; D011994:Recombinant Proteins / Q000528:radiation effects; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared; D014466:Ultraviolet Rays
D001597:Benzylidene Compounds; D008164:Luminescent Proteins; D010860:Pigments, Biological; D011522:Protons; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins