Inhibition of heat-induced protein aggregation by zirconium phthalocyanines.

Specific proteins found in food sources tend to aggregate into fibrils under heat treatment; studying these aggregation processes and developing tools to control protein heat-induced aggregation is an active area of research. Phthalocyanine complexes are known to exhibit antiprionic and anti-fibrillogenic activity. Thus, the anti-fibrillogenic effect of a series of Zr phthalocyanines with different out-of-plane coordinated ligands, namely positively charged (PcZrLys2 ), negatively charged (PcZrCitr2 ), and group able to form disulfide bridges (PcZrS2 ), on the heat-induced aggregation of such proteins as BLG, insulin, and lysozyme was studied. The inhibition of reaction activity up to about 90% was observed in the presence of these compounds for all proteins. The effective concentration of the inhibitor was calculated for the compound with the highest activity (PcZrS2 ) to be 10.6 ± 3.6 and 7.3 ± 1.2 µM/L, respectively. Fluorescence spectroscopy studies demonstrated similar binding constants of three phthalocyanines binding with BLG globule. This is consistent with the results of molecular dynamics simulation, which imply the interaction of the globule with the tetrapyrrole macrocycle of phthalocyanine, leading to the globule stabilization. At the same time, TEM shows that in the presence of phthalocyanine PcZrS2 , thinner and longer fibrils were formed compared to control in all three proteins (BLG, insulin, and lysozyme). Thus, we can conclude that phthalocyanine PcZrS2 affects the amyloid aggregation's general mechanism, which is typical for proteins of different structures. Therefore, the phthalocyanine PcZrS2 is proposed as an anti-amyloidogenic agent suppressing heat-induced aggregation of proteins of different structures, making it potentially suitable for application in the food industry.

3D-Shaped Binders of Unfolded Proteins Inducing Cancer Cell-Specific Endoplasmic Reticulum Stress In Vitro and In Vivo.

The amount of unfolded proteins is increased in cancer cells, leading to endoplasmic reticulum (ER) stress. Therefore, cancer cells are sensitive to drugs capable of further enhancing ER stress. Examples of such drugs include the clinically approved proteosome inhibitors bortezomib and carfilzomib. Unfortunately, the known ER stress inducers exhibit dose-limiting side effects that justify the search for better, more cancer-specific drugs of this type. Herein, we report on FeC 2, which binds to unfolded proteins prevents their further processing, thereby leading to ER stress and ROS increase in cancer cells, but not in normal cells. FeC 2 exhibits low micromolar toxicity toward human acute promyelocytic leukemia HL-60, Burkitt's lymphoma BL-2, T-cell leukemia Jurkat, ovarian carcinoma A2780, lung cancer SK-MES-1, and murine lung cancer LLC1 cells. Due to the cancer-specific mode of action, 2 is not toxic in vivo up to the dose of 147 mg/kg, does not affect normal blood and bone marrow cells at the therapeutically active dose, but strongly suppresses both primary tumor growth (confirmed in Nemeth-Kellner lymphoma and LLC1 lung cancer models of murine tumor) and spreading of metastases (LLC1).

Study of tetraphenylporphyrins as modifiers of insulin amyloid aggregation.

Amyloid fibrils are rigid ß-pleated protein aggregates that are connected with series of harmful diseases and at the same time are promising as base for novel nanomaterials. Thus, design of compounds able to inhibit or redirect those aggregates formation is important both for the biomedical aims and for nanotechnology applications. Here, we studied the effect of tetraphenylporphyrins (metal free, their Cu and Pd complexes, and those functionalized by carboxy and amino groups on periphery) on insulin amyloid self-assembling. The strongest impact on insulin aggregation was demonstrated by a metal-free porphyrin bearing four carboxy groups. This compound strongly suppresses insulin aggregation (about 88% reduction in amyloid-sensitive probe emission) inducing formation of fibrils with the length close to this of free insulin (1.7 ± 0.6 µm as compared with 1.4 ± 0.4 µm, respectively) with an essentially reduced tendency to lateral aggregation. Contrarily, the presence of tetraphenylporphyrin containing four amino groups only slightly affects fibrils' morphology and makes weaker impact on insulin aggregation yield (about 44% reduction). This is explained by the ability of aromatic carboxy groups of 5,10,15,20-(tetra-4-carboxyphenyl)porphyrin to interact with complementary protein-binding groups and thus stabilize the supramolecular complex. For 5,10,15,20-(tetra-4-aminophenyl)porphyrin, full protonation takes place in acidic medium of protein aggregation reaction; this results in the high positive charge of TPPN4 (equal or close to +6) and hence higher contribution of coulombic repulsion to interaction of TPPN4 with insulin. One more possible mechanism of the lower inhibition effect of TPPN4 as compared with TPPC4 could be the more restricted possibility of the former as compared with the latter to form H bonds with insulin groups. It was also shown that metal-free, Pd-containing, and Cu-containing tetraphenylporphyrins without peripheral substituents make almost the same impact on the protein self-assembling. We suppose this to be due to coordination saturation of these metal atoms.

Cyclodextrin Encapsulated pH Sensitive Dyes as Fluorescent Cellular Probes: Self-Aggregation and In Vitro Assessments.

We have designed and synthesized a series of novel, supramolecular, long-lived fluorescent probes based on the host-guest inclusion complexes formation between fluorescent indolizinyl-pyridinium salts and ß-cyclodextrin. Fluorescence and electrospray ionisation mass spectrometry experiments, supported by theoretical molecular docking studies, were utilized in the monitoring of the inclusion complexes formation, evidencing the appearance of corresponding 1:1 and 1:2 species. Additionally, the influence of the guest molecule over the aggregation processes of the cyclodextrin inclusion complexes was investigated by transmission electron microscopy. The absence of cytotoxicity, cellular permeability, long-lived intracellular fluorescence, and in time specific accumulation within acidic organelles identified the investigated supramolecular entities as remarkable candidates for intracellular fluorescence probes. Co-staining experiments using specific organelle markers revealed the fact that, after a 24-h incubation period, the inclusion complexes accumulate predominantly in lysosomes rather than in mitochondria. This study opens new possibilities for a broad range of fluorescent dyes with solubility and high toxicity issues, able to form inclusion complexes with ß-cyclodextrin, to be tested as intracellular fluorescence probes.

The impact of binding of macrocyclic metal complexes on amyloid fibrillization of insulin and lysozyme.

Amyloid fibrils are insoluble protein aggregates whose accumulation in cells and tissues is connected with a range of pathological diseases. We studied the impact of 2 metal complexes (axially coordinated Hf phthalocyanine and iron (II) clathrochelate) on aggregation of insulin and lysozyme. For both proteins, the host-guest interaction with these compounds changes the kinetics of fibrillization and affects the morphology of final aggregates. The Hf phthalocyanine is a very efficient inhibitor of insulin fibrillization; in its presence, only very low amounts of fibrils with the diameters of 0.8 to 5 nm and spherical aggregates were found. Effective concentration of fibrillization inhibition (IC50 ) was estimated to be 0.11 ± 0.04 µM. The clathrochelate induced the formation of thin fibrils with the diameters of 0.8 to 2.5 nm; IC50 was estimated as 20 ± 9 µM. The lysozyme fibrillization remained quite intensive in the presence of the studied compounds; they induced the formation of long filaments (the length up to 2.5 µm, the diameters of 1.5-3.5 nm). These fibrils noticeably differed from those of free lysozyme short linear species (the diameters of 3-5 nm, the length up to 0.6 µm). Thinning and elongation of fibrils suggest that the metal complexes bind mainly to the grooves of protofilaments; this hinders the stacking of early aggregates or protofilaments together but does not hinder their growth. The image of the fibril separated into 2 protofilaments allows suggesting that the fibril formation occurs via the growth of the parallel protofilaments with their subsequent twisting in the fibril. The changes of the lysozyme intrinsic fluorescence indicate that both metal complexes interact with the protein during the stage of the fibrillar seeds formation.

Cytotoxicity of electrophilic iron(II)-clathrochelates in human promyelocytic leukemia cell line.

We observed that electrophilic iron(II)-clathrochelates exhibit significant cytotoxicity in human promyelocytic leukemia cells (IC50=6.5±4.6µM), which correlates with the enhancement of intracellular oxidative stress (17-fold increase with respect to the cells treated with the solvent only). Based on in vitro studies we suggested that this effect is caused by alkylation of glutathione leading to inhibition of the cellular antioxidative system and by catalytic generation of reactive oxygen species by products of the alkylation reaction.

Trimethine cyanine dyes as fluorescent probes for amyloid fibrils: The effect of N,N'-substituents.

The effect of various N,N'-substituents in the molecule of benzothiazole trimethine cyanine dye on its ability to sense the amyloid aggregates of protein was studied. The dyes are low fluorescent when free and in the presence of monomeric proteins, but their emission intensity sharply increases in complexes with aggregated insulin and lysozyme, with the fluorescence quantum yield reaching up to 0.42. The dyes carrying butyl, hydroxyalkyl, and phenylalkyl groups as N,N'-substituents possess the increased fluorescent sensitivity to fibrillar lysozyme, whereas the ones carrying quaternary amino groups are preferably sensitive to fibrillar insulin. This fluorescent sensitivity preference provided by the N,N'-functional groups could be explained by the interaction between these groups and protein side chains. The strongest fluorescent response (up to 70times) and the same sensitivity to aggregates of both proteins were exhibited by the dye D-51 carrying N-sulfoalkyl group. The studied cyanines allow the detection of fibrillar aggregates in the wide range up to 0.8 to 300µg/ml and permit monitoring the protein aggregation kinetics with high reproducibility. The modification of trimethine cyanine dyes by functional substituents in N,N'-positions is suggested as a tool for the design of fluorescent molecules with the enhanced fluorescent sensitivity to the fibrillar aggregates of proteins.

Study of anti-fibrillogenic activity of iron(II) clathrochelates.

The macrocyclic compounds mono- and bis-iron(II) clathrochelates were firstly studied as potential anti-fibrillogenic agents using fluorescent inhibitory assay, atomic force microscopy and flow cytometry. It is shown that presence of the clathrochelates leads to the change in kinetics of insulin fibrillization reaction and reduces the amount of formed fibrils (up to 70%). The nature of ribbed substituent could determine the activity of clathrochelates-the higher inhibitory effect is observed for compounds containing carboxybenzenesulfide groups, while the inhibitory properties only slightly depend on the size of complex species. The mono- and bis-clathrochelate derivatives of meta-mercaptobenzoic acid have close values of IC50 namely 16 ± 2 and 24 ± 5 µM, respectively. The presence of clathrochelates decreases the fibril diameter from 5-12 nm for free insulin fibrils to 3-8 nm for these formed in the clathrochelate presence, it also prevents the lateral aggregation of mature fibrils and formation of superfibrillar clusters. However the addition of clathrochelate results in more heterogeneous (both by size and structure) insulin aggregates population as compared to the free insulin. This way, cage complexes-iron(II) clathrochelates are proposed as efficient agents able to suppress the protein aggregation processes.

Interaction of the iron(II) cage complexes with proteins: protein fluorescence quenching study.

Interaction of the iron(II) mono- and bis-clathrochelates with bovine serum albumin (BSA), ß-lactoglobulin, lysozyme and insulin was studied by the steady-state and time-resolved fluorescent spectroscopies. These cage complexes do not make significant impact on fluorescent properties of ß-lactoglobulin, lysozyme and insulin. At the same time, the monoclathrochelates strongly quench a fluorescence intensity of BSA and substantially decrease its excited state lifetime due to their binding to this protein. This occurs due to the excitation energy transfer from a tryptophan residue to a cage molecule or/and to the change of the tryptophan nearest environment caused by either clathrochelate binding or an alteration of the BSA conformation. The effect of the iron(II) bis-clathrochelate on BSA fluorescence is much weaker as compared to its monomacrobicyclic analogs as a result of an increase in its size.

Studies of anti-fibrillogenic activity of phthalocyanines of zirconium containing out-of-plane ligands.

Series of phthalocyanines of zirconium containing lysine, citric, nonanoic acid residues and dibenzolylmethane groups as out-of-plane ligands are firstly studied as inhibitors of fibrillogenesis using cyanine-based fluorescent inhibitory assay. It was shown that studied phthalocyanines at concentration of 20µM inhibited aggregation reaction on 38.5-57.6% and inhibitory activity of phthalocyanines depended on the chemical nature of out-of-plane ligand. For the most active compound PcZrLys(2) (zirconium phthalocyanine containing lysine fragment) the efficient inhibitor concentration was estimated to be 37µM. AFM studies have shown that in the presence of PcZrLys(2) the inhibition of fibrils formation and formation of spherical oligomeric aggregates took place. Due to the ability of phthalocyanines to decrease efficiently protein aggregation into the amyloid fibrils, modification of phthalocyanine molecules via out-of-plane substitutions was proposed as approach for design of anti-fibrillogenic agents with required properties.

Trimethine Cyanine Dyes as NA-Sensitive Probes for Visualization of Cell Compartments in Fluorescence Microscopy.

We propose symmetrical cationic trimethine cyanine dyes with ß-substituents in the polymethine chain based on modified benzothiazole and benzoxazole heterocycles as probes for the detection and visualization of live and fixed cells by fluorescence microscopy. The spectral-luminescent properties of trimethine cyanines have been characterized for free dyes and in the presence of nucleic acids (NA) and globular proteins. The studied cyanines are low to moderate fluorescent when free, but in the presence of NA, they show an increase in emission intensity up to 111 times; the most pronounced emission increase was observed for the dyes T-2 in the presence of dsDNA and T-1 with RNA. Spectral methods showed the binding of all dyes to nucleic acids, and different interaction mechanisms have been proposed. The ability to visualize cell components of the studied dyes has been evaluated using different human cell lines (MCF-7, A2780, HeLa, and Hs27). We have shown that all dyes are cell-permeant staining nucleus components, probably RNA-rich nucleoli with background fluorescence in the cytoplasm, except for the dye T-5. The dye T-5 selectively stains some structures in the cytoplasm of MCF-7 and A2780 cells associated with mitochondria or lysosomes. This effect has also been confirmed for the normal type of cell line-human foreskin fibroblasts (Hs27). The costaining of dye T-5 with MitoTracker CMXRos Red demonstrates specificity to mitochondria at a concentration of 0.1 µM. Colocalization analysis has shown signals overlapping of dye T-5 and MitoTracker CMXRos Red (Pearson's Coefficient value = 0.92 ± 0.04). The photostability study shows benzoxazole dyes to be up to ∼7 times more photostable than benzothiazole ones. Moreover, studied benzoxazoles are less cytotoxic at working concentrations than benzothiazoles (67% of cell viability for T-4, T-5 compared to 12% for T-1, and ∼30% for T-2, T-3 after 24 h). Therefore, the benzoxazole T-4 dye is proposed for nucleic acid detection in vitro and intracellular fluorescence imaging of live and fixed cells. In contrast, the benzoxazole dye T-5 is proposed as a good alternative to commercial dyes for mitochondria staining in the green-yellow region of the spectrum.

Mono and trimethine cyanines Cyan 40 and Cyan 2 as probes for highly selective fluorescent detection of non-canonical DNA structures.

Two of earlier reported dsDNA sensitive cyanine dyes-monomethine Cyan 40 and meso-substituted trimethine Cyan 2 were studied for their ability to interact with non-canonical DNA conformations. These dyes were characterized by spectral-luminescent methods in the presence of G-quadruplex, triplex and dsDNA motifs. We have demonstrated that Cyan 2 binds strongly and preferentially to triple- and quadruple-stranded DNA forms that results in a strong enhancement of the dye fluorescence, as compared to dsDNA, while Cyan 40 form fluorescent complexes preferentially only with the triplex form. Highly fluorescent complexes of Cyan 2 with DNA triplexes and G-quadruplexes and Cyan 40 with DNA triplexes are very stable and do not dissociate during gel electrophoresis, leading to preferential staining of the above DNA forms in gels. The data presented point to the intercalation mechanism of the Cyan 2 binding to G4-DNA, while the complexes of Cyan 40 and Cyan 2 with triplex DNA are believed to be formed via groove binding mode. The Cyan dyes can provide a highly sensitive method for detection and quantification of non-canonical structures in genome.

Hydroxy and methoxy substituted thiacarbocyanines for fluorescent detection of amyloid formations.

In present paper series of trimethine cyanines modified in 5,5'- or 6,6'- position with hydroxy- or methoxy- substituents is studied for their ability to interact selectively with fibrillar formations. Processes of dye aggregation that accompany this interaction were also investigated. Meso-methyl trimethynecyanines with 5,5'- methoxy (7519) and hydroxy (7515) substituents strongly (up to 40 times) increase fluorescence intensity in the presence of fibrillar insulin, and also give noticeable fluorescent response on the presence of various aggregated proteins (lysozyme, ß-lactoglobulin, α-synuclein A53T). 7519 and 7515 dyes can be used for fluorometric detection of fibrillar insulin at concentrations of approximately 1.5-120 microg/ml. For meso-ethyl substituted dye 7514 the ability to form H- and J-aggregates upon interaction with insulin fibrils was suggested. The model of the H- and J-aggregate packing in the protein fibrillar structure has been proposed.

Modification of insulin amyloid aggregation by Zr phthalocyanines functionalized with dehydroacetic acid derivatives.

Amyloid fibrils are widely studied both as target in conformational disorders and as basis for the development of protein-based functional materials. The three Zr phthalocyanines bearing dehydroacetic acid residue (PcZr(L1)2) and its condensed derivatives (PcZr(L2)2 and PcZr(L3)2) as out-of-plane ligands were synthesized and their influence on insulin fibril formation was studied by amyloid-sensitive fluorescent dye based assay, scanning electron microscopy, fluorescent and absorption spectroscopies. The presence of Zr phthalocyanines was shown to modify the fibril formation. The morphology of fibrils formed in the presence of the Zr phthalocyanines differs from that of free insulin and depends on the structure of out-of-plane ligands. It is shown that free insulin mostly forms fibril clusters with the length of about 0.3-2.1 µm. The presence of Zr phthalocyanines leads to the formation of individual 0.4-2.8 µm-long fibrils with a reduced tendency to lateral aggregation and cluster formation (PcZr(L1)2), shorter 0.2-1.5 µm-long fibrils with the tendency to lateral aggregation without clusters (PcZr(L2)2), and fibril-like 0.2-1.0 µm-long structures (PcZr(L3)2). The strongest influence on fibrils morphology made by PcZr(L3)2 could be explained by the additional stacking of phenyl moiety of the ligand with aromatic amino acids in protein. The evidences of binding of studied Zr phthalocyanines to mature fibrils were shown by absorption spectroscopy (for PcZr(L1)2 and PcZr(L2)2) and fluorescent spectroscopy (for PcZr(L3)2). These complexes could be potentially used as external tools allowing the development of functional materials on protein fibrils basis.

Synthesis and spectral characterization of the first fluorescein-tagged iron(ii) clathrochelates, their supramolecular interactions with globular proteins, and cellular uptake.

A fluorescein-tagged iron(ii) cage complex was obtained in a moderate total yield using a two-step synthetic procedure starting from its propargylamine-containing clathrochelate precursor. An 11-fold decrease in fluorescence quantum yield is observed in passing from the given fluorescein-based dye to its clathrochelate derivative. An excitation energy transfer from the terminal fluorescent group of the macrobicyclic molecule to its quasiaromatic highly π-conjugated clathrochelate framework can explain this effect. The kinetics of the hydrolysis of the acetyl groups of acetylated fluorescein azide and its clathrochelate derivative in the presence of one equivalent of BSA evidenced no strong supramolecular host-guest interactions between BSA and the tested compounds. Study of a chemical stability of the deacetylated iron(ii) clathrochelate suggested the formation of a supramolecular 1 : 1 BSA-clathrochelate assembly. Moreover, an addition of BSA or HSA to its solution caused the appearance of strong clathrochelate-based ICD outputs. The fluorescence emission anisotropy studies also evidenced the supramolecular binding of the fluorescein-tagged iron(ii) clathrochelate to the BSA macromolecule, leading to a high increase in this type of anisotropy. Subcellular uptake of the fluorescein-tagged molecules was visualized using fluorescence microscopy and showed its distribution to be mainly in the cytosol without entering the nucleus or accumulating in any other organelle. An X-rayed crystal of the above propargylamide macrobicyclic precursor with a reactive terminal C[triple bond, length as m-dash]C bond contains the clathrochelate molecules of two types, A and B. The encapsulated iron(ii) ion in these molecules is situated in the center of its FeN6-coordination polyhedron, the geometry of which is intermediate between a trigonal prism (TP) and a trigonal antiprism (TAP). The Fe-N distances vary from 1.8754(6) to 1.9286(4) Å and the heights h of their distorted TP-TAP polyhedra are very similar (2.30 and 2.31 Å); their values of φ are equal to 25.3 and 26.6°. In this crystal, the molecules of types A and B participate in different types of hydrogen bonding, giving H-bonded clathrochelate tetramers through their carboxylic and amide groups, respectively; these tetramers are connected to H-bonded chains.

Monomethine cyanine probes for visualization of cellular RNA by fluorescence microscopy.

We have studied spectral-luminescent properties of the monomethine cyanine dyes both in their free states and in the presence of either double-stranded deoxyribonucleic acids (dsDNAs) or single-stranded ribonucleic acids (RNAs). The dyes possess low fluorescence intensity in an unbound state, which is increased up to 479 times in the presence of the nucleic acids. In the presence of RNAs, the fluorescence intensity increase was stronger than that observed in the presence of dsDNA. Next, we have performed staining of live and fixed cells by all prepared dyes. The dyes proved to be cell and nuclear membrane permeant. They are photostable and brightly stain RNA-containing organelles in both live and fixed cells. The colocalization confirmed the specific nucleoli staining with anti-Ki-67 antibodies. The RNA digestion experiment has confirmed the selectivity of the dyes toward intracellular RNA. Based on the obtained results, we can conclude that the investigated monomethine cyanine dyes are useful fluorescent probes for the visualization of intracellular RNA and RNA-containing organelles such as nucleoli by using fluorescence microscopy.

Styryl dyes as two-photon excited fluorescent probes for DNA detection and two-photon laser scanning fluorescence microscopy of living cells.

Spectral-fluorescent properties of benzothiazole styryl monomer (Bos-3) and homodimer (DBos-21) dyes in presence of DNA were studied. The dyes enhance their fluorescence intensity in 2-3 orders of magnitude upon interaction with DNA. Studied styrylcyanines in DNA presence demonstrate rather high values of two-photon absorption (TPA) cross-section, which are comparable with the values of TPA cross section of the rhodamine dyes. An applicability of the styrylcyanines as probes for the fluorescence microscopy of living cells was studied. It was shown that both dyes are cell-permeable but homodimer dye DBos-21 produces noticeably brighter staining of HeLa cells comparing with monomer dye Bos-3. Molecules of DBos-21 initially bind to the nucleic acids-containing cell organelles (presumable mitochondria) and are able to penetrate into the cell nucleus. Thus, homodimer styryl DBos-21 dye is viewed as efficient stain for single-photon and two-photon excitation fluorescence imaging of living cells.

Studies of interaction between cyanine dye T-284 and fibrillar alpha-synuclein.

A key feature of Parkinson's disease is the formation and accumulation of amyloid fibrils of the natively unfolded protein α-synuclein (ASN) inside neurons. Recently we have proposed novel sensitive monomethinecyanine dye T-284 as fluorescent probe for quantitative detection of ASN amyloid fibrils. In this study the T-284 dye complex with ASN fibril was characterized by means of fluorescence anisotropy, atomic force microscopy and time-resolved fluorescence techniques to give further insights into the mode of dye interaction with amyloid fibrils. The fluorescence anisotropy of T-284 was shown to noticeably increase upon addition of aggregated proteins indicating on stable dye/amyloid fibril complex formation. AFM imaging of fibrillar wild-type ASN revealed differences in heights between ASN fibrils alone and in presence of the T-284 dye (6.37 ± 1.0 nm and 8.0 ± 1.1 nm respectively), that is believed to be caused by embedding of T-284 dye molecules in the "binding channel" running along the fibril. Fluorescence decay analysis of the T-284 in complexes with fibrillar ASN variants revealed the fluorescence lifetime values for T-284/fibril complexes to be an order of magnitude higher as compared to the free dye. Also, the fluorescence decay of free T-284 was bi-exponential, while dye bound to protein yields tri-exponential decay. We suppose that in complexes with fibrillar ASN variants T-284 dye might exist in different "populations" due to interaction with fibrils in different conformers and ways. The exact binding mode of T-284 with ASN fibrils needs further studies. Studied parameters of dye/amyloid fibril complexes are important for the characterization and screening of newly-developed amyloid-sensitive dyes.

Fluorescent ß-ketoenole AmyGreen dye for visualization of amyloid components of bacterial biofilms.

Green-emitting water-soluble amino-ketoenole dye AmyGreen is proposed as an efficient fluorescent stain for visualization of bacterial amyloids in biofilms and the detection of pathological amyloids in vitro. This dye is almost non-fluorescent in solution, displays strong green emission in the presence of amyloid fibril of proteins. AmyGreen is also weakly fluorescent in presence to biomolecules that are components of cells, extracellular matrix or medium: nucleic acids, polysaccharides, lipids, and proteins. Thus, the luminescence turn-on behavior of AmyGreen can be utilized for visualization of amyloid components of bacterial biofilm extracellular matrix. Herein we report the application of AmyGreen for fluorescent staining of a number of amyloid-contained bacteria biofilms produced by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bordetella avium, and Staphylococcus aureus. The effectiveness of AmyGreen was compared to traditional amyloid sensitive dye Thioflavine T. The main advantage of AmyGreen (concentration 10-5 M) is a higher sensitivity in the visualization of amyloid biofilm components over Thioflavine T (10-4 M) as it was revealed when staining E. coli and K. pneumoniae bacterial biofilms. Besides, AmyGreen displays lower cross-selectivity to nucleic acids as demonstrated both in in-solution experiments and upon staining of eukaryotic human mesenchymal stem cells used as amyloid-free negative control over amyloid-rich bacterial biofilms. The results point to a lower risk of false-positive response upon determination of amyloid components of bacterial biofilm using AmyGreen. Co-staining of biofilm by AmyGreen and cellulose sensitive dye Calcofluor White show difference in their staining patterns and localization, indicating separation of polysaccharide-rich and amyloid-rich regions of investigated biofilms. Thus, we suggest the new AmyGreen stain for visualization and differentiation of amyloid fibrils in bacterial biofilms to be used solely and in combination with other stains for confocal and fluorescence microscopy analysis.

Sensing of Proteins by ICD Response of Iron(II) Clathrochelates Functionalized by Carboxyalkylsulfide Groups.

Recognition of elements of protein tertiary structure is crucial for biotechnological and biomedical tasks; this makes the development of optical sensors for certain protein surface elements important. Herein, we demonstrated the ability of iron(II) clathrochelates (1-3) functionalized with mono-, di- and hexa-carboxyalkylsulfide to induce selective circular dichroism (CD) response upon binding to globular proteins. Thus, inherently CD-silent clathrochelates revealed selective inducing of CD spectra when binding to human serum albumin (HSA) (1, 2), beta-lactoglobuline (2) and bovine serum albumin (BSA) (3). Hence, functionalization of iron(II) clathrochelates with the carboxyalkylsulfide group appears to be a promising tool for the design of CD-probes sensitive to certain surface elements of proteins tertiary structure. Additionally, interaction of 1-3 with proteins was also studied by isothermal titration calorimetry, protein fluorescence quenching, electrospray ionization mass spectrometry (ESI-MS) and computer simulations. Formation of both 1:1 and 1:2 assemblies of HSA with 1-3 was evidenced by ESI-MS. A protein fluorescence quenching study suggests that 3 binds with both BSA and HSA via the sites close to Trp residues. Molecular docking calculations indicate that for both BSA and HSA, binding of 3 to Site I and to an "additional site" is more favorable energetically than binding to Site II.