Structural and spectral response of Aequorea victoria green fluorescent proteins to chromophore fluorination.
Global 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.
Pal
Prajna P
PP
Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany.
Bae
Jae Hyun
JH
Azim
M Kamran
MK
Hess
Petra
P
Friedrich
Rainer
R
Huber
Robert
R
Moroder
Luis
L
Budisa
Nediljko
N
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D005462:Fluorine Radioisotopes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D037402:Hydrozoa; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D005462:Fluorine Radioisotopes; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; C001245:3-fluorotyrosine; D049452:Green Fluorescent Proteins; D014443:Tyrosine; C060489:2-fluorotyrosine