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

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journal contribution
posted on 18.12.2015, 00:00 by Deng-Hsiang Chang, Chun-Lin Ou, Hung-Yu Hsu, Guan-Jhih Huang, Chen-Yi Kao, Yi-Hung Liu, Shie-Ming Peng, Eric Wei-Guang Diau, Jye-Shane Yang
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.