Nitric Oxide Sensing by a Blue Fluorescent Protein.

S-Nitrosylation of cysteine residues is an important molecular mechanism for dynamic, post-translational regulation of several proteins, providing a ubiquitous redox regulation. Cys residues are present in several fluorescent proteins (FP), including members of the family of Aequorea victoria Green Fluorescent Protein (GFP)-derived FPs, where two highly conserved cysteine residues contribute to a favorable environment for the autocatalytic chromophore formation reaction. The effect of nitric oxide on the fluorescence properties of FPs has not been investigated thus far, despite the tremendous role FPs have played for 25 years as tools in cell biology. We have examined the response to nitric oxide of fluorescence emission by the blue-emitting fluorescent protein mTagBFP2. To our surprise, upon exposure to micromolar concentrations of nitric oxide, we observed a roughly 30% reduction in fluorescence quantum yield and lifetime. Recovery of fluorescence emission is observed after treatment with Na-dithionite. Experiments on related fluorescent proteins from different families show similar nitric oxide sensitivity of their fluorescence. We correlate the effect with S-nitrosylation of Cys residues. Mutation of Cys residues in mTagBFP2 removes its nitric oxide sensitivity. Similarly, fluorescent proteins devoid of Cys residues are insensitive to nitric oxide. We finally show that mTagBFP2 can sense exogenously generated nitric oxide when expressed in a living mammalian cell. We propose mTagBFP2 as the starting point for a new class of genetically encoded nitric oxide sensors based on fluorescence lifetime imaging.

Enhanced photosensitizing properties of protein bound curcumin.

AIMS: The naturally occurring compound curcumin has been proposed for a number of pharmacological applications. In spite of the promising chemotherapeutic properties of the molecule, the use of curcumin has been largely limited by its chemical instability in water. In this work, we propose the use of water soluble proteins to overcome this issue in perspective applications to photodynamic therapy of tumors. MATERIALS AND METHODS: Curcumin was bound to bovine serum albumin and its photophysical properties was studied as well as its effect on cell viability after light exposure through MTT assay and confocal imaging. KEY FINDINGS: Bovine serum albumin binds curcumin with moderate affinity and solubilizes the hydrophobic compound preserving its photophysical properties for several hours. Cell viability assays demonstrate that when bound to serum albumin, curcumin is an effective photosensitizer for HeLa cells, with better performance than curcumin alone. Confocal fluorescence imaging reveals that when curcumin is delivered alone, it preferentially associates with mitochondria, whereas curcumin bound to bovine serum albumin is found in additional locations within the cell, a fact that may be related to the higher phototoxicity observed in this case. SIGNIFICANCE: The higher bioavailability of the photosensitizing compound curcumin when bound to serum albumin may be exploited to increase the efficiency of the drug in photodynamic therapy of tumors.

Apomyoglobin is an efficient carrier for zinc phthalocyanine in photodynamic therapy of tumors.

The spectral and the photophysical properties of phthalocyanines have made these dyes attractive for applications in photodynamic therapy of cancer. One important known issue of these compounds is their tendency to aggregate in aqueous media, which decreases their fluorescence, triplet, and singlet oxygen quantum yields. We report on the use of apomyoglobin as a carrier for zinc phthalocyanine (ZnPc) to overcome solubility limitations of the dye. We show that the protein is able to bind ZnPc in monomeric form, preserving its photophysics. Confocal fluorescence imaging of PC3 and HeLa cells, treated with the complex between ZnPc and apomyoglobin, demonstrates that the photosensitizer is uptaken quickly by cells. Illumination of treated cells strongly decreases viability, as demonstrated by live/dead fluorescence assay.

Electrostatic Tuning of the Ligand Binding Mechanism by Glu27 in Nitrophorin 7.

Nitrophorins (NP) 1-7 are NO-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. The isoform NP7 displays peculiar properties, such as an abnormally high isoelectric point, the ability to bind negatively charged membranes, and a strong pH sensitivity of NO affinity. A unique trait of NP7 is the presence of Glu in position 27, which is occupied by Val in other NPs. Glu27 appears to be important for tuning the heme properties, but its influence on the pH-dependent NO release mechanism, which is assisted by a conformational change in the AB loop, remains unexplored. Here, in order to gain insight into the functional role of Glu27, we examine the effect of Glu27 → Val and Glu27 → Gln mutations on the ligand binding kinetics using CO as a model. The results reveal that annihilation of the negative charge of Glu27 upon mutation reduces the pH sensitivity of the ligand binding rate, a process that in turn depends on the ionization of Asp32. We propose that Glu27 exerts a through-space electrostatic action on Asp32, which shifts the pKa of the latter amino acid towards more acidic values thus reducing the pH sensitivity of the transition between open and closed states.

Zinc-Substituted Myoglobin Is a Naturally Occurring Photo-antimicrobial Agent with Potential Applications in Food Decontamination.

Zinc-substituted myoglobin (ZnMb) is a naturally occurring photosensitizer that generates singlet oxygen with a high quantum yield. Using a combination of photophysical and fluorescence imaging techniques, we demonstrate the interaction of ZnMb with Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. An efficient antibacterial action against S. aureus was observed, with a reduction up to 99.9999% in the number of colony-forming units, whereas no sizable effect was detected against E. coli. Because ZnMb is known to form during the maturation of additive-free not-cooked cured ham, the use of this protein as a built-in photodynamic agent may constitute a viable method for the decontamination of these food products from Gram-positive bacteria.