Green fluorescent protein ground states: the influence of a second protonation site near the chromophore.
The photophysical properties of most green fluorescent protein mutants (GFPs) are strongly affected by pH. This effect must be carefully taken into account when using GFPs as fluorescent probes or indicators. Usually, the pH-dependence of GFPs is rationalized on the basis of the ionization equilibrium of the chromophore phenol group. Yet many different mutants show spectral behavior that cannot be explained by ionization of this group alone. In this study, we propose a general model of protonation comprising two ionization sites (2S model). Steady-state optical measurements at different pH and temperature and pH-jump relaxation experiments were combined to highlight the thermodynamic and kinetic properties of paradigmatically different GFP variants. Our experiments support the 2S model. For the case of mutants in which E222 is the second protonation site, thermodynamic coupling between this residue's and the chromophore's ionization reactions was demonstrated. In agreement with the 2S model predictions, X-ray analysis of one of these mutants showed the presence of two chromophore populations at high pH.
Bizzarri
Ranieri
R
Scuola Normale Superiore-IIT Research Unit, Piazza dei Cavalieri 7 I-56126 Pisa, Italy. r.bizzarri@ sns.it
Nifosì
Riccardo
R
Abbruzzetti
Stefania
S
Rocchia
Walter
W
Guidi
Sara
S
Arosio
Daniele
D
Garau
Gianpiero
G
Campanini
Barbara
B
Grandi
Elena
E
Ricci
Fernanda
F
Viappiani
Cristiano
C
Beltram
Fabio
F
D019943:Amino Acid Substitution / Q000235:genetics; D018360:Crystallography, X-Ray; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D010777:Photochemistry; D011522:Protons; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D011522:Protons; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins