bi0484825_si_001.pdf (511.98 kB)
Structural and Spectral Response of Aequorea victoria Green Fluorescent Proteins to Chromophore Fluorination†
journal contribution
posted on 2005-03-15, 00:00 authored by Prajna P. Pal, Jae Hyun Bae, M. Kamran Azim, Petra Hess, Rainer Friedrich, Robert Huber, Luis Moroder, Nediljko BudisaGlobal replacements of tyrosine by 2- and 3-fluorotyrosine in “enhanced green” and “enhanced
yellow” mutants of Aequorea victoria green fluorescent proteins (avGFPs) provided protein variants with
novel biophysical properties. While crystallographic and modeled structures of these proteins are
indistinguishable from those of their native counterparts (i.e., they are perfectly isomorphous), there are
considerable differences in their spectroscopic properties. The fluorine being an integral part of the avGFP
chromophore induces changes in the titration curves, variations in the intensity of the absorbance and
fluorescence, and spectral shifts in the emission maxima. Furthermore, targeted fluorination in close
proximity to the fluorinated chromophore yielded additional variants with considerably enhanced spectral
changes. These unique spectral properties are intrinsic features of the fluorinated avGFPs, in the context
of the rigid chromophore−microenvironment interactions. The availability of the isomorpohous crystal
structures of fluorinated avGFPs allowed mapping of novel, unusual interaction distances created by the
presence of fluorine atoms. In addition, fluorine atoms in the ortho position of the chromophore tyrosyl
moiety exhibit a single conformation, while in the meta position two conformer states were observed in
the crystalline state. Such global replacements in chromophores of avGFPs and similar proteins result in
“atomic mutations” (i.e., H → F replacements) in the structures, offering unprecedented opportunities to
understand and manipulate the relationships between protein structure and spectroscopic properties.