Cooperativity and Site-Selectivity of Intramolecular Hydrogen Bonds on the Fluorescence Quenching of Modified GFP Chromophores

This paper provides the first example
of experimentally characterized
hydrogen-bond cooperativity on fluorescence quenching with a modified
green fluorescence protein (GFP) chromophore that contains a 6-membered
CN···H–O and a 7-membered CO···H–O
intramolecular H-bonds. Variable-temperature 1H NMR and
electronic absorption and emission spectroscopies were used to elucidate
the preference of intra- vs intermolecular H-bonding at different
concentrations (1 mM and 10 μM), and X-ray crystal structures
provide clues of possible intermolecular H-bonding modes. In the ground
state, the 6-membered H-bond is significant but the 7-membered one
is rather weak. However, fluorescence quenching is dominated by the
7-membered H-bond, indicating a strengthening of the H-bond in the
excited state. The H-bonding effect is more pronounced in more polar
solvents, and no intermediates were observed from femtosecond fluorescence
decays. The fluorescence quenching is attributed to the occurrence
of diabatic excited-state proton transfer. Cooperativity of the two
intramolecular H-bonds on spectral shifts and fluorescence quenching
is evidenced by comparing with both the single H-bonded and the non-H-bonded
counterparts. The H-bond cooperativity does not belong to the conventional
patterns of σ- and π-cooperativity but a new type of polarization
interactions, which demonstrates the significant interplay of H-bonds
for multiple H-bonding systems in the electronically excited states.