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Visualization of differential gene expression by improved cyan fluorescent protein and yellow fluorescent protein production in Bacillus subtilis.

ABSTRACT: The distinguishable cyan and yellow fluorescent proteins (CFP and YFP) enable the simultaneous in vivo visualization of different promoter activities. Here, we report new cloning vectors for the construction of cfp and yfp fusions in Bacillus subtilis. By extending the N-terminal portions of previously described CFP and YFP variants, 20- to 70-fold-improved fluorescent-protein production was achieved. Probably, the addition of sequences encoding the first eight amino acids of the N-terminal part of ComGA of B. subtilis overcomes the slow translation initiation that is provoked by the eukaryotic codon bias present in the original cfp and yfp genes. Using these new vectors, we demonstrate that, within an isogenic population of sporulating B. subtilis cells, expression of the abrB and spoIIA genes is distinct in individual cells.

SUBMITTER: Veening JW

PROVIDER: S-EPMC525234 | biostudies-literature | 2004 Nov

REPOSITORIES: biostudies-literature

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Visualization of differential gene expression by improved cyan fluorescent protein and yellow fluorescent protein production in Bacillus subtilis.

Veening Jan-Willem JW Smits Wiep Klaas WK Hamoen Leendert W LW Jongbloed Jan D H JD Kuipers Oscar P OP

Applied and environmental microbiology 20041101 11

The distinguishable cyan and yellow fluorescent proteins (CFP and YFP) enable the simultaneous in vivo visualization of different promoter activities. Here, we report new cloning vectors for the construction of cfp and yfp fusions in Bacillus subtilis. By extending the N-terminal portions of previously described CFP and YFP variants, 20- to 70-fold-improved fluorescent-protein production was achieved. Probably, the addition of sequences encoding the first eight amino acids of the N-terminal part ...[more]

PMID: 15528548

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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.

Dataset Information

Visualization of differential gene expression by improved cyan fluorescent protein and yellow fluorescent protein production in Bacillus subtilis.

Publications

Visualization of differential gene expression by improved cyan fluorescent protein and yellow fluorescent protein production in Bacillus subtilis.

Similar Datasets

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