Engineered Metal Binding Sites on Green Fluorescence Protein (original) (raw)
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Possible Application of Metal Sensitive Red Fluorescent Proteins in Environmental Monitoring
The metal sensitivity and selectivity of two fluorescent proteins was investigated. The studied proteins are mutant forms of the red fluorescent protein (DsRed); according to our results, both proteins showed sensitivity to the presence of copper[II] and nickel ions. The fluorescence intensity of the proteins has decreased significantly in the presence of copper ions in the micromolar range. Metal binding by these proteins is a reversible process, as addition of a chelating agent liberates the bound ions. Considering these advantageous properties, the studied proteins can be used as copper ion biosensors.
Metal-enhanced fluorescence: an emerging tool in biotechnology
Current Opinion in Biotechnology, 2005
Over the past 15 years, fluorescence has become the dominant detection/sensing technology in medical diagnostics and biotechnology. Although fluorescence is a highly sensitive technique, where single molecules can readily be detected, there is still a drive for reduced detection limits. The detection of a fluorophore is usually limited by its quantum yield, autofluorescence of the samples and/or the photostability of the fluorophores; however, there has been a recent explosion in the use of metallic nanostructures to favorably modify the spectral properties of fluorophores and to alleviate some of these fluorophore photophysical constraints. The use of fluorophore-metal interactions has been termed radiative decay engineering, metal-enhanced fluorescence or surface-enhanced fluorescence.
Over the past 15 years, fluorescence has become the dominant detection/sensing technology in medical diagnostics and biotechnology. Although fluorescence is a highly sensitive technique, where single molecules can readily be detected, there is still a drive for reduced detection limits. The detection of a fluorophore is usually limited by its quantum yield, autofluorescence of the samples and/or the photostability of the fluorophores; however, there has been a recent explosion in the use of metallic nanostructures to favorably modify the spectral properties of fluorophores and to alleviate some of these fluorophore photophysical constraints. The use of fluorophore–metal interactions has been termed radiative decay engineering, metal-enhanced fluorescence or surface-enhanced fluorescence.
Binding of chimeric metal-binding green fluorescent protein to lipid monolayer
European Biophysics …, 2004
Membrane-based bioanalytical devices for metal determination using green fluorescent protein as the sensor molecule may be a useful future biomimetic material. However, in order to develop such a device, it is necessary first to understand the interaction of the protein with lipid membranes. Thus we have investigated the interaction between chimeric cadmium-binding green fluorescent proteins (CdBPGFPs) and lipid monolayers, using a film-balance technique complemented with epifluorescence microscopy. The binding avidity was monitored from the surface pressure vs. area isotherms or from the measured increase in the lateral pressure upon injection of the chimeric CdBPGFPs beneath the lipid monolayer. Increased fluidization as well as expansion of the surface area were shown to depend on the concentration of the CdBPGFPs. The kinetics of the protein-induced increase in lateral pressure was found to be biphasic. The chimeric CdBPGFPs possessed high affinity to the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer with a dissociation constant of K d =10 )8 M. Epifluorescence measurements showed that this affinity is due to the presence of the Cd-binding peptide, which caused the GFP to incorporate preferentially to the liquid phase and defect part of the rigid domain at low interfacial pressure. At high compression, the Cd-binding peptide could neither incorporate nor remain in the lipid core. However, specific orientation of the chimeric CdBPGFPs underneath the air-water interface was achieved, even under high surface pressure, when the proteins were applied to the metal-chelating lipidcontaining surfaces. This specific binding could be controlled reversibly by the addition of metal ions or metal chelator. The reversible binding of the chimeric CdBPGFPs to metal-chelating lipids provided a potential approach for immobilization, orientation and lateral organization of a protein at the membrane interface. Furthermore, the feasibility of applying the chelator lipids for the codetermination of metal ions with specific ligands was also revealed. Our finding clearly demonstrates that a strong interaction, particularly with fluid lipid domains, could potentially be used for sensor development in the future.
Proceedings - Society of Photo-Optical Instrumentation Engineers, 2004
We describe the development of a novel generic approach to fluorescence sensing based on metal-enhanced fluorescence (MEF). This work follows our initial reports of radiative decay engineering (RDE), where we experimentally demonstrated dramatic signal enhancements of fluorophores positioned close to surface-bound silver nanostructures. The attractive changes in spectral properties of fluorophores includes increased rates of excitation, increased quantum yields, decreased fluorescence lifetimes with an increased photostability, and drastically increased rates of multi-photon excitation. In this report we present a new class of fluorescent biomarkers which are strongly enhanced by metallic particles. This has afforded the development of a novel generic approach for ultra-sensitive fluorescence assay technology. The assay platform utilizes metal particles deposited on glass/quartz surfaces, covered with sub-nanometer layers of a fluorescent biomarker. As such the fluorescence signal o...
Journal of Fluorescence, 2013
Two histidines were introduced by sitedirected mutagenesis into the structure of Enhanced Green Fluorescent Protein, replacing the serine at position 202 and the glutamine at position 204 for increasing the sensitivity of the protein towards different metal ions by creating possible metal binding sites near the chromophore group. There is no appreciable difference between the absorbance and fluorescence spectra of the two proteins (wild type and the double-histidine mutant) indicating that the mutation does not change the environment of the fluorophore. Fluorescence quenching was measured at different pH (6.5-8) and temperatures (20-45°C) varying the concentration of metal ions. Under optimal conditions (pH07.5, 20°C) the mutant's K d is 16 nM, it binds copper more than 200fold stronger than the wild type EGFP.
Structural chemistry of a green fluorescent protein Zn biosensor
Zinc metalloproteins influence DNA synthesis, microtubule polymerization, gene expression, apoptosis, and immune system function. 1 Furthermore, Zn(II) homeostasis is critical for complex neurobiological systems: Zn(II) acts in synaptic transmission, as a contributory factor in neurological disorders including epilepsy and Alzheimer's disease, and as a neurotoxin under seizure or pathological conditions. 1 To fully understand Zn(II) functions in cell and neurobiology, probes are needed that are capable of monitoring in vivo spatial and temporal distributions of metal ions. Designed small molecule Zn(II) fluorescent probes have many advantages. 2 However, protein-based biosensors 3 might complement these probes as proteins can be optimized for selectivity and affinity, added noninvasively to cells by transfection, and targeted to specific tissues, organelles, or cellular locations. Recently, proteins have been engineered to incorporate desired properties via structurebased 4 and directed evolution 5 strategies. Since protein metal site specificity and activity are exquisitely sensitive to precise ligand chemistry and geometry, structure-based design of metal ion biosensors would benefit from very high resolution structures of apo and distinct metal-bound protein states. We report here (i) the design and characterization of a green fluorescent protein (GFP) mutant that binds Zn(II) (enhancing fluorescence intensity) and Cu-(II) (quenching fluorescence) to a tridentate chromophore resembling half a porphyrin and (ii) very high-resolution crystallographic structures for these apo, Zn(II)-bound, and Cu(II)-bound proteins ( ). These structures, reported with experimentally determined standard uncertainty errors, define accurate metal-site geometric parameters for a novel GFP metal-binding site and provide critical structural chemistry for understanding the metal ion affinity, specificity, and activity of protein metal sites. Moreover, these results prompt structure-based hypotheses to explain the protein's metal ion selectivity, metal binding kinetics, and fluorescence enhancement upon Zn(II) binding, with potential general implications for the design of protein metal ion biosensors.
Fluorescent Protein-Based Optical Biosensor for Copper Ion Quantitation
… trace element research, 2010
In the present study, spectroscopic determinations of copper ions using chimeric metal-binding green fluorescent protein (His6GFP) as an active indicator have been explored. Supplementation of copper ions to the GFP solution led to a remarkable decrease of fluorescent intensity corresponding to metal concentrations. For circumstances, rapid declining of fluorescence up to 60% was detected in the presence of 500 μM copper. This is in contrast to those observed in the case of zinc and calcium ions, in which approximately 10-20% of fluorescence was affected. Recovery of its original fluorescence up to 80% was mediated by the addition of ethylenediamine tetraacetic acid. More importantly, in the presence of metal ions, the emission wavelength maximum remains unchanged while reduction of the optical density of the absorption spectrum has been observed. This indicates that the chromophore's ground state was possibly affected by the static quenching process. Results from circular dichroism measurements revealed that the overall patterns of circular dichroism spectra after exposure to copper ions were not significantly different from that of the control, where the majority of sharp positive band around 195-196 nm in combination with a broad negative deflection around 215-216 nm was obtained. Taken together, it can be presumed that copper ions exerted their static quenching on the fluorescence rather than structural or conformational alteration. However, notification has to be made that some peptide rearrangements may also occur in the presence of metal ions. Further studies were conducted to investigate the feasibility of using the His6GFP as a sensing unit for copper ions. The His6GFP was encapsulated in Sol-gel and immobilized onto the optical fiber connected with a fluorescence detecting device. The Sol-gel was doped into the metal solution where the quenching of fluorescence could be monitored in real time. The sensing unit provided a high sensitivity of detection in the range of 0.5 μM to
Biomedical Vibrational Spectroscopy and Biohazard Detection Technologies, 2004
We describe the development of a novel generic approach to fluorescence sensing based on metalenhanced fluorescence (MEF). This work follows our initial reports of radiative decay engineering (RDE), where we experimentally demonstrated dramatic signal enhancements of fluorophores positioned close to surface-bound silver nanostructures. The attractive changes in spectral properties of fluorophores includes increased rates of excitation, increased quantum yields, decreased fluorescence lifetimes with an increased photostability, and drastically increased rates of multiphoton excitation. In this report we present a new class of fluorescent biomarkers which are strongly enhanced by metallic particles. This has afforded the development of a novel generic approach for ultra-sensitive fluorescence assay technology. The assay platform utilizes metal particles deposited on glass/quartz surfaces, covered with sub-nanometer layers of a fluorescent biomarker. As such the fluorescence signal of the composite is strongly enhanced. This readily allows easy, quantitative and inexpensive fluorescence detection of minimal traces of specific antigens. We also explore different sensing geometries, such as using evanescent wave excitation.