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PH-dependent optical properties of synthetic fluorescent imidazoles.

ABSTRACT: An imidazole moiety is often found as an integral part of fluorophores in a variety of fluorescent proteins and many such proteins display pH-dependent light emission. In contrast, synthetic fluorescent compounds with incorporated imidazoles are rare and have not been studied as pH probes. In this report, the richness of imidazole optical properties, including pH sensitivity, was demonstrated by means of a novel imidazole-based fluorophore 1H-imidazol-5-yl-vinylbenz[e]indolium. Three species corresponding to protonated, neutral, and deprotonated imidazoles were identified in the broad range of pH 1-12. The absorption and emission bands of each species were assigned by comparative spectral analysis with synthesized mono- and di-N-methylated fluorescent imidazole analogues. pK(a) analysis in the ground and the excited states showed photoacidic properties of the fluorescent imidazoles due to the excited state proton transfer (ESPT). This effect was negligible for substituted imidazoles. The assessment of a pH-sensitive center in the imidazole ring revealed the switching of the pH-sensitive centers from 1-N in the ground state to 3-N in the excited state. The effect was attributed to the unique kind of the excited state charge transfer (ESCT) resulting in a positive charge swapping between two nitrogens.

SUBMITTER: Berezin MY

PROVIDER: S-EPMC2844652 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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pH-dependent optical properties of synthetic fluorescent imidazoles.

Berezin Mikhail Y MY Kao Jeff J Achilefu Samuel S

Chemistry (Weinheim an der Bergstrasse, Germany) 20090101 14

An imidazole moiety is often found as an integral part of fluorophores in a variety of fluorescent proteins and many such proteins display pH-dependent light emission. In contrast, synthetic fluorescent compounds with incorporated imidazoles are rare and have not been studied as pH probes. In this report, the richness of imidazole optical properties, including pH sensitivity, was demonstrated by means of a novel imidazole-based fluorophore 1H-imidazol-5-yl-vinylbenz[e]indolium. Three species cor ...[more]

PMID: 19212987

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Project description:A recently engineered mutant of cyan fluorescent protein (WasCFP) that exhibits pH-dependent absorption suggests that its tryptophan-based chromophore switches between neutral (protonated) and charged (deprotonated) states depending on external pH. At pH 8.1, the latter gives rise to green fluorescence as opposed to the cyan color of emission that is characteristic for the neutral form at low pH. Given the high energy cost of deprotonating the tryptophan at the indole nitrogen, this behavior is puzzling, even if the stabilizing effect of the V61K mutation in proximity to the protonation/deprotonation site is considered. Because of its potential to open new avenues for the development of optical sensors and photoconvertible fluorescent proteins, a mechanistic understanding of how the charged state in WasCFP can possibly be stabilized is thus important. Attributed to the dynamic nature of proteins, such understanding often requires knowledge of the various conformations adopted, including transiently populated conformational states. Transient conformational states triggered by pH are of emerging interest and have been shown to be important whenever ionizable groups interact with hydrophobic environments. Using a combination of the weighted-ensemble sampling method and explicit-solvent constant pH molecular dynamics (CPHMD(MS?D)) simulations, we have identified a solvated transient state, characterized by a partially open ?-barrel where the chromophore pKa of 6.8 is shifted by over 20 units from that of the closed form (6.8 and 31.7, respectively). This state contributes a small population at low pH (12% at pH 6.1) but becomes dominant at mildly basic conditions, contributing as much as 53% at pH 8.1. This pH-dependent population shift between neutral (at pH 6.1) and charged (at pH 8.1) forms is thus responsible for the observed absorption behavior of WasCFP. Our findings demonstrate the conditions necessary to stabilize the charged state of the WasCFP chromophore (namely, local solvation at the deprotonation site and a partial flexibility of the protein ?-barrel structure) and provide the first evidence that transient conformational states can control optical properties of fluorescent proteins.

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PH-dependent optical properties of synthetic fluorescent imidazoles.

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pH-dependent optical properties of synthetic fluorescent imidazoles.

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