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X-ray diffraction analysis and molecular-replacement solution of the cyan fluorescent protein dsFP483.

ABSTRACT: A novel cyan fluorescent protein, dsFP483 from the coral Discosoma striata, has been crystallized. Diffraction data were collected to 2.1 A and processed in space group C2. Molecular-replacement methods were applied using the closely related red fluorescent protein DsRed as a search model. The asymmetric unit appears to contain six protein molecules (1.5 tetramers), five of which (83%) could be located by the molecular-replacement searches.

SUBMITTER: Wang M

PROVIDER: S-EPMC1991309 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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X-ray diffraction analysis and molecular-replacement solution of the cyan fluorescent protein dsFP483.

Wang Meitian M Patel Hetal N HN Wachter Rebekka M RM

Acta crystallographica. Section F, Structural biology and crystallization communications 20050930 Pt 10

A novel cyan fluorescent protein, dsFP483 from the coral Discosoma striata, has been crystallized. Diffraction data were collected to 2.1 A and processed in space group C2. Molecular-replacement methods were applied using the closely related red fluorescent protein DsRed as a search model. The asymmetric unit appears to contain six protein molecules (1.5 tetramers), five of which (83%) could be located by the molecular-replacement searches. ...[more]

PMID: 16511196

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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

X-ray diffraction analysis and molecular-replacement solution of the cyan fluorescent protein dsFP483.

Publications

X-ray diffraction analysis and molecular-replacement solution of the cyan fluorescent protein dsFP483.

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