title: Green fluorescent protein ground states: the influence of a second protonation site near the chromophore.
abstract: 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.
authors: Scuola Normale Superiore-IIT Research Unit, Piazza dei Cavalieri 7 I-56126 Pisa, Italy. r.bizzarri@ sns.it
authors: Ranieri
authors: R
authors: Bizzarri
authors: Riccardo
authors: R
authors: Nifosì
authors: Stefania
authors: S
authors: Abbruzzetti
authors: Walter
authors: W
authors: Rocchia
authors: Sara
authors: S
authors: Guidi
authors: Daniele
authors: D
authors: Arosio
authors: Gianpiero
authors: G
authors: Garau
authors: Barbara
authors: B
authors: Campanini
authors: Elena
authors: E
authors: Grandi
authors: Fernanda
authors: F
authors: Ricci
authors: Cristiano
authors: C
authors: Viappiani
authors: Fabio
authors: F
authors: Beltram
mesh_terms: 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
chemical_list: D011522:Protons; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins