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Excited state proton transfer in the red fluorescent protein mKeima.

ABSTRACT: mKeima is an unusual monomeric red fluorescent protein (lambda(em)(max) approximately 620 nm) that is maximally excited in the blue (lambda(ex)(max) approximately 440 nm). The large Stokes shift suggests that the chromophore is normally protonated. A 1.63 A resolution structure of mKeima reveals the chromophore to be imbedded in a novel hydrogen bond network, different than in GFP, which could support proton transfer from the chromophore hydroxyl, via Ser142, to Asp157. At low temperatures the emission contains a green component (lambda(em)(max) approximately 535 nm), enhanced by deuterium substitution, presumably resulting from reduced proton transfer efficiency. Ultrafast pump/probe studies reveal a rising component in the 610 nm emission with a lifetime of approximately 4 ps, characterizing the rate of proton transfer. Mutation of Asp157 to neutral Asn changes the chromophore resting charge state to anionic (lambda(ex)(max) approximately 565 nm, lambda(em)(max) approximately 620 nm). Thus, excited state proton transfer (ESPT) explains the large Stokes shift. This work unambiguously characterizes green emission from the protonated acylimine chromophore of red fluorescent proteins.

SUBMITTER: Henderson JN

PROVIDER: S-EPMC2754865 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Excited state proton transfer in the red fluorescent protein mKeima.

Henderson J Nathan JN Osborn Maire F MF Koon Nayden N Gepshtein Rinat R Huppert Dan D Remington S James SJ

Journal of the American Chemical Society 20090901 37

mKeima is an unusual monomeric red fluorescent protein (lambda(em)(max) approximately 620 nm) that is maximally excited in the blue (lambda(ex)(max) approximately 440 nm). The large Stokes shift suggests that the chromophore is normally protonated. A 1.63 A resolution structure of mKeima reveals the chromophore to be imbedded in a novel hydrogen bond network, different than in GFP, which could support proton transfer from the chromophore hydroxyl, via Ser142, to Asp157. At low temperatures the e ...[more]

PMID: 19708654

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Project description:Fluorescent probes that exhibit solvatochromic or excited-state proton-transfer (ESPT) properties are essential tools for the study of complex biological or chemical systems. Herein, the synthesis and characterization of a novel fluorophore that reveals both features, 5-isocyanonaphthalene-1-ol (ICOL), are reported. Various solvatochromic methods, such as Lippert-Mataga and Bilot-Kawski, together with time-dependent density functional theory (TD-DFT) and time-resolved emission spectroscopy (TRES), were applied to gain insights into its excited-state behavior. To make comparisons, the octyloxy derivative of ICOL, 5-isocyano-1-(octyloxy)naphthalene (ICON), was also prepared. We found that internal charge transfer (ICT) takes place between the isocyano and -OH groups of ICOL, and we determined the values of the dipole moments for the ground and excited states of both ICOL and ICON. Furthermore, in the emission spectra of ICOL, a second band at higher wavelengths (green emission) in solvents of higher polarities (dual emission), in addition to the band present at lower wavelengths (blue emission), were observed. The extent of this dual emission increases in the order of 2-propanol < methanol < N,N-dimethylformamide (DMF) < dimethyl sulfoxide (DMSO). The presence of the dual fluorescence of ICOL in these solvents can be ascribed to ESPT. For ICOL, we also determined ground- and excited-state pKa values of 8.4 ± 0.3 and 0.9 ± 0.7, respectively, which indicates a considerable increase in acidity upon excitation. The TRES experiments showed that the excited-state lifetimes of the ICOL and ICON spanned from 10.1 ns to 5.0 ns and from 5.7 ns to 3.8 ns, respectively. In addition, we demonstrated that ICOL can be used as an effective indicator of not only the critical micelle concentration (cmc) of ionic (sodium lauryl sulfate (SLS)) and nonionic surfactants (Tween 80), but also other micellar parameters, such as partition coefficients, as well as to map the microenvironments in the cavities of biomacromolecules (e.g., BSA). It is also pointed out that fluorescence quenching by pyridine can effectively be utilized for the determination of the fractions of ICOL molecules that reside at the water-micelle interface and in the interior spaces of micelles.

Dataset Information

Excited state proton transfer in the red fluorescent protein mKeima.

Publications

Excited state proton transfer in the red fluorescent protein mKeima.

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