Three-step Förster resonance energy transfer on an amyloid fibril scaffold.

The present study provides evidence that the energy transfer chain consisting of the benzothiazole dye Thioflavin T as an input donor, a phosphonium dye TDV and a squaraine dye SQ4 as mediators, and one of the three squaraines SQ1/2/3 as an output acceptor displays an excellent amyloid-sensing ability when applied to differentiating between the amyloid and non-fibrillized states of insulin. The ensemble of fluorophores offers the advantages of a large effective Stokes shift (∼240 nm), well-resolved 3D fluorescence patterns and strong enhancement of the terminal fluorescence (up to two orders of magnitude). The occurrence of multistep energy transfer on an amyloid fibril scaffold opens new possibilities for the more sensitive detection of fibrillar protein assemblies and their applications in nanophotonics.

Probing the interactions of novel europium coordination complexes with serum albumin.

Molecular interactions between novel europium coordination complexes (EC) possessing superior cytotoxic activity and bovine serum albumin (BSA), the most prominent representative of plasma proteins, were assessed using fluorescence spectroscopy and molecular docking techniques. Cumulative results from fluorescent probe binding, fluorescence quenching and Förster resonance energy transfer studies revealed that the europium complexes V4 and V8 do not perturb the BSA structure, while V3, V5, and V7 induce partial unfolding of the polypeptide chain. Molecular docking studies coupled with analysis of the three-dimensional structure of the BSA-EC complexes showed that V4 and V8 reside in the vicinity of the protein IIA subdomain (Sudlow's site I), while V3, V5 and V5 were localized predominantly in the BSA IIIA subdomain (Sudlow's site II). Due to the intactness of the protein structure upon association with V4 and V8, these compounds may be recommended for further evaluation as potential antineoplastic agents.

Lipid Bilayer Interactions of Amyloidogenic N-Terminal Fragment of Apolipoprotein A-I Probed by Förster Resonance Energy Transfer and Molecular Dynamics Simulations.

The effects of one of the amyloidogenic mutations of apolipoprotein A-I (apoA-I), G26R, on the thermal stability, structural dynamics and lipid-associating properties of the 1-83 N-terminal fragment of apoA-I (A83) have been investigated using the Förster resonance energy transfer (FRET) and molecular dynamics (MD) simulation. The measurements of FRET between the tryptophan residues of the single Trp variants of A83 as donors and the membrane-incorporated fluorescent probe 4-dimethylaminochalcone as an acceptor provided evidence for a less depth of A83/G26R penetration into phosphatidylcholine (PC) bilayer compared to WT counterpart. The unfolding MD simulations showed that G26R mutation destabilizes the overall structure of A83, with individual alpha-helices differing in their thermal stability. The MD simulations performed at physiological temperature revealed that A83 and A83/G26R differ in their conformational behavior in an aqueous solution, PC and PC/Cholesterol bilayers. These findings may prove of importance for deeper understanding of the key determinants of apoA-I amyloidogenesis.

Förster Resonance Energy Transfer Study of Cytochrome c-Lipid Interactions.

Specific interactions between a mitochondrial hemoprotein cytochrome c (cyt c) and cardiolipin, a lipid component of mitochondrial membrane, are crucial to electron shuttling and apoptotic activities of this protein. In the present study the Förster resonance energy transfer (FRET) between anthrylvinyl-labeled phosphatidylcholine as a donor and heme moiety of cyt c as an acceptor was employed to give a quantitative characterization of the protein binding to the model membranes from the mixtures of phosphatidylcholine (PC) with phosphatidylglycerol (PG), phosphatidylserine (PS) or cardiolipin (CL) in different molar ratios. The multiple arrays of the FRET data were globally analyzed in terms of the model of energy transfer in two-dimensional systems combined with the scaled particle adsorption model. The arguments in favor of the specificity of cyt c interactions with CL were obtained, including the higher adsorption potential and the deeper protein insertion in the lipid bilayer.

Liposomal Co-Encapsulation of Two Novel Europium Complexes and Doxorubicin: Fluorescence Study.

The present study was undertaken to design the novel liposomal drug formulation containing doxorubicin and europium coordination complexes. It was shown that co-encapsulation of the drugs facilitates the partitioning and permeation of lanthanides into the lipid bilayer. The obtained results suggest that new drug platform may have potential application in the design of novel antitumor agents.

Symmetric Meso-Chloro-Substituted Pentamethine Cyanine Dyes Containing Benzothiazolyl/Benzoselenazolyl Chromophores Novel Synthetic Approach and Studies on Photophysical Properties upon Interaction with bio-Objects.

A series of symmetric pentamethine cyanine dyes derived from various N-substituted benzothiazolium/benzoselenazolium salts, and a conjugated bis-aniline derivative containing a chlorine atom at meso-position with respect to the polymethine chain, were synthesized using a novel improved synthetic approach under mild conditions at room temperature. The reaction procedure was held by grinding the starting compounds for relative short times. The novel method is reliable and highly reproducible. Some photophysical characteristics were recorded in various solvents, including absorption, and fluorescence quantum yields using Cy-5 as a reference. Additional studies on interactions with several bio-objects such as liposomes, DNA, and proteins have been investigated in the present work.

FRET evidence for untwisting of amyloid fibrils on the surface of model membranes.

Apolipoprotein A-I (apoA-I) is an amyloid-forming protein whose amyloidogenic properties are attributed mainly to its N-terminal fragment. Cell membranes are thought to be the primary target for the toxic amyloid aggregates. In the present study Förster resonance energy transfer (FRET) between the membrane fluorescent probe Laurdan as a donor and amyloid-specific dye Thioflavin T (ThT) as an acceptor was employed to explore the interactions of amyloid fibrils from apoA-I variants 1-83/G26R and 1-83/G26R/W@8 with the model membranes composed of phosphatidylcholine and its mixture with cholesterol. The changes in FRET efficiency upon fibril-lipid binding were found to correlate with the extent of protein fibrillization. AFM imaging revealed the presence of two polymorphic states of fibrillar 1-83/G26R/W@8 with the helical and twisted ribbon morphologies. The simulation-based analysis of the experimental FRET profiles provided the arguments in favor of untwisting of fibrillar assemblies upon their interaction with the model membranes. Evidence for the face-on orientation and superficial bilayer location of the membrane-bound fragments of 1-83/G26R/W@8 fibrils was obtained.

Membrane effects of N-terminal fragment of apolipoprotein A-I: a fluorescent probe study.

The binding of monomeric and aggregated variants of 1-83 N-terminal fragment of apolipoprotein A-I with substitution mutations G26R, G26R/W@8, G26R/W@50 and G26R/W@72 to the model lipid membranes composed of phosphatidylcholine and its mixture with cholesterol has been investigated using fluorescent probes pyrene and Laurdan. Examination of pyrene spectral behavior did not reveal any marked influence of apoA-I mutants on the hydrocarbon region of lipid bilayer. In contrast, probing the membrane effects by Laurdan revealed decrease in the probe generalized polarization in the presence of aggregated proteins. suggesting that oligomeric and fibrillar apoA-I species induce increase in hydration degree and reduction of lipid packing density in the membrane interfacial region. These findings may shed light on molecular details of amyloid cytotoxicity.

Interactions of Lipid Membranes with Fibrillar Protein Aggregates.

Amyloid fibrils are an intriguing class of protein aggregates with distinct physicochemical, structural and morphological properties. They display peculiar membrane-binding behavior, thus adding complexity to the problem of protein-lipid interactions. The consensus that emerged during the past decade is that amyloid cytotoxicity arises from a continuum of cross-ß-sheet assemblies including mature fibrils. Based on literature survey and our own data, in this chapter we address several aspects of fibril-lipid interactions, including (i) the effects of amyloid assemblies on molecular organization of lipid bilayer; (ii) competition between fibrillar and monomeric membrane-associating proteins for binding to the lipid surface; and (iii) the effects of lipids on the structural morphology of fibrillar aggregates. To illustrate some of the processes occurring in fibril-lipid systems, we present and analyze fluorescence data reporting on lipid bilayer interactions with fibrillar lysozyme and with the N-terminal 83-residue fragment of amyloidogenic mutant apolipoprotein A-I, 1-83/G26R/W@8. The results help understand possible mechanisms of interaction and mutual remodeling of amyloid fibers and lipid membranes, which may contribute to amyloid cytotoxicity.

Interaction of thioflavin T with amyloid fibrils of apolipoprotein A-I N-terminal fragment: resonance energy transfer study.

Apolipoprotein A-I is amenable to a number of specific mutations associated with hereditary systemic amyloidoses. Amyloidogenic properties of apoA-I are determined mainly by its N-terminal fragment. In the present study Förster resonance energy transfer between tryptophan as a donor and Thioflavin T as an acceptor was employed to obtain structural information on the amyloid fibrils formed by apoA-I variant 1-83/G26R/W@8. Analysis of the dye-fibril binding data provided evidence for the presence of two types of ThT binding sites with similar stoichiometries (bound dye to monomeric protein molar ratio ∼10), but different association constants (∼6 and 0.1µM(-1)) and ThT quantum yields in fibril-associated state (0.08 and 0.05, respectively). A ß-strand-loop-ß-strand structural model of 1-83/G26R/W@8 apoA-I fibrils has been proposed, with potential ThT binding sites located in the solvent-exposed grooves of the N-terminal ß-sheet layer. Reasoning from the expanded FRET analysis allowing for heterogeneity of ThT binding centers and fibril polymorphism, the most probable locations of high- and low-affinity ThT binding sites were attributed to the grooves T16_Y18 and D20_L22, respectively.

Fluorescence Investigation of Interactions Between Novel Benzanthrone Dyes and Lysozyme Amyloid Fibrils.

A series of novel fluorescent benzanthrone dyes have been tested for their ability to identify and characterize fibrillar aggregates of lysozyme prepared by protein denaturation in concentrated ethanol solution (F(eth)) or acidic buffer (F(ac)). Quantitative parameters of the dye association with native and fibrillar protein have been derived from the results of fluorimetric titration. The binding characteristics proved to be different for F(eth)- and F(ac)-bound benzanthrones, highlighting the dye sensitivity to the distinctions in fibril morphology. By comparing the dye preference to fibrillar protein aggregates, AM2, A8 and A6 were selected as the most prospective amyloid tracers. Based on the analysis of red edge excitation shifts and fluorescence lifetimes of the amyloid-bound dyes it was assumed that surface grooves or dry "steric zipper" interface are potential fibril binding sites for the novel fluorophores.

Location of novel benzanthrone dyes in model membranes as revealed by resonance energy transfer.

Förster resonance energy transfer (FRET) between anthrylvinyl-labeled phosphatidylcholine (AV-PC) as a donor and newly synthesized benzanthrones (referred to here as A8, A6, AM12, AM15 and AM18) as acceptors has been examined to gain insight into molecular level details of the interactions between benzanthrone dyes and model lipid membranes composed of zwitterionic lipid phosphatidylcholine and its mixtures with anionic lipids cardiolipin (CL) and phosphatidylglycerol (PG). FRET data were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for A8 location in phospholipid headgroup region has been obtained. Inclusion of CL and PG into PC bilayer has been found to induce substantial relocation of A6, AM12, AM15 and AM18 from hydrophobic membrane core to lipid-water interface.

Radiolabeling of bionanomaterials with technetium 99m: current state and future prospects.

Radiolabeling of bionanomaterials with technetium-99m (99mTc) has become a promising approach in combining the benefits of nanotechnology and nuclear medicine for diagnostic and therapeutic purposes. This review is intended to provide a comprehensive overview of the state-of-the-art of radiolabeling of bionanomaterials with 99mTc, highlighting the synthesis methods, labeling mechanisms, biological evaluation, physicochemical characterization and clinical applications of 99mTc-labeled bionanomaterials. Various types of nanomaterials are considered in the review, including lipid- and protein-based nanosystems, dendrimers and polymeric nanomaterials. Moreover, the review assesses the challenges presented by this emerging field, such as stability of the radiolabel, potential toxicity of the nanomaterials and regulatory aspects. Finally, promising future perspectives and areas of research development in 99mTc-labeled bionanomaterials are discussed.

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Europium coordination complexes as potential anticancer drugs: their partitioning and permeation into lipid bilayers as revealed by pyrene fluorescence quenching.

The present study was undertaken to evaluate the membrane-associating properties of a series of novel antitumor agents, Eu(III) coordination complexes (EC), using the pyrene fluorescence quenching as an analytical instrument. Analysis of EC-induced decrease in pyrene fluorescence intensity in terms of partition and solubility-diffusion models allowed us to evaluate the partition and permeation coefficients of the examined compounds into the lipid vesicles prepared from zwitterionic lipid phosphatidylcholine (PC) and its mixtures with cholesterol (Chol) and anionic lipid cardiolipin (CL). The drug-lipid interactions were found to have the complex nature determined by both EC structure and lipid bilayer composition. High values of the obtained partition and permeation coefficients create the background for the development of EC liposomal formulations.

Fluorescence study of the membrane effects of aggregated lysozyme.

The last decade has seen unprecedented upsurge of interest in the structural and toxic properties of particular type of protein aggregates, amyloid fibrils, associated with a number of pathological states. In the present study fluorescence spectroscopy technique has been employed to gain further insight into the membrane-related mechanisms of amyloid toxicity. To this end, erythrocyte model system composed of liposomes and hemoglobin was subjected to the action of oligomeric and fibrillar lysozyme. Acrylamide quenching of lysozyme fluorescence showed that solvent accessibility of Trp62 and Trp108 increases upon the protein fibrillization. Resonance energy transfer measurements suggested the possibility of direct complexation between hemoglobin and aggregated lysozyme. Using the novel squaraine dye SQ-1 it was demonstrated that aggregated lysozyme is capable of inhibiting lipid peroxidation processes. Fluorescent probes pyrene, Prodan and diphenylhexatriene were employed to characterize the membrane-modifying properties of hemoglobin and lysozyme. Both oligomeric and fibrillar forms of lysozyme were found to exert condensing influence on lipid bilayer structure, with the membrane effects of fibrils being less amenable to modulation by hemoglobin.

Deciphering the molecular details of interactions between anti-COVID drugs and functional human proteins: in silico approach.

The molecular docking calculations have been employed to investigate the interactions a set of proteins with the repurposed anti-COVID drugs. The position of the therapeutic agents within the protein structure was dependent on a particular drug-protein system and varied from the binding cleft to the periphery of the polypeptide chain. Interactions involved in the drug-protein complexation includes predominantly hydrogen bonding and hydrophobic contacts. The obtained results may be of particular importance while developing the anti-COVID strategies as well as for deeper understanding of the drug pharmacodynamics and pharmacokinetics.

Novel benzanthrone aminoderivatives for membrane studies.

The potential of novel benzanthrone aminoderivatives to trace the changes in physicochemical properties of lipid bilayer has been evaluated. Binding of the dyes to the lipid bilayers composed of zwitterionic phospholipid phosphatidylcholine (PC) and its mixtures with anionic phospholipid cardiolipin (CL) and cholesterol (Chol) was followed by significant quantum yield increase with small blue shift of emission maximum. Analysis of partition coefficients of the dyes under study showed that all aminobenzanthrones possess high lipid-associating ability. The dyes A8 and AM2 proved to be sensitive to the variations in membrane chemical composition responding to the changes in bilayer hydration induced by CL and Chol.

FÓ§rster resonance energy transfer analysis of amyloid state of proteins.

The Förster resonance energy transfer (FRET) is a well-established and versatile spectroscopic technique extensively used for exploring a variety of biomolecular interactions and processes. The present review is intended to cover the main results of our FRET studies focused on amyloid fibrils, a particular type of disease-associated protein aggregates. Based on the examples of several fibril-forming proteins including insulin, lysozyme and amyloidogenic variants of N-terminal fragment of apolipoprotein A-I, it was demonstrated that: (i) the two- and three-step FRET with the classical amyloid marker Thioflavin T as an input donor has a high amyloid-sensing potential and can be used to refine the amyloid detection assays; (ii) the intermolecular time-resolved and single-molecule pulse interleaved excitation FRET can give quantitative information on the nucleation of amyloid fibrils; (iii) FRET between the membrane fluorescent probes and protein-associated intrinsic or extrinsic fluorophores is suitable for monitoring the membrane binding of fibrillar proteins, exploring their location relative to lipid-water interface and restructuring on a lipid matrix; (iv) the FRET-based distance estimation between fibril-bound donor and acceptor fluorophores can serve as one of the verification criteria upon structural modeling of amyloid fibrils.

Fluorescence spectroscopy of protein oligomerization in membranes.

Fluorescence spectroscopy is one of the most powerful tools for characterization of a multitude of biological processes. Of these, the phenomenon of protein oligomerization attracts especial interest due to its crucial role in the formation of fibrillar protein aggregates (amyloid fibrils) involved in ethiology of so-called protein misfolding diseases. It is becoming increasingly substantiated that protein fibrillization in vivo can be initiated and modulated at membrane-water interface. All steps of membrane-assisted fibrillogenesis, viz., protein adsorption onto lipid bilayer, structural transition of polypeptide chain into a highly aggregation-prone partially folded conformation, assembly of oligomeric nucleus from membrane-bound monomeric species and fiber elongation can be monitored with a mighty family of fluorescence-based techniques. Furthermore, the mechanisms behind cytotoxicity of prefibrillar protein oligomers are highly amenable to fluorescence analysis. The applications of fluorescence spectroscopy to monitoring protein oligomerization in a membrane environment are exemplified and some problems encountered in such kinds of studies are highlighted.

Fluorescence study of lipid bilayer interactions of Eu(III) coordination complexes.

The interaction between Eu(III) tris-ß-diketonato coordination complexes (EC), displaying antitumor activity, and lipid vesicles composed of zwitterionic lipid phosphatidylcholine has been studied using fluorescence spectroscopy techniques. To characterize EC-membrane binding, several fluorescent probes, including pyrene, Prodan and 1,6-diphenyl-1,3,5-hexatriene, have been employed. It has been found that EC display effective partitioning into lipid phase, giving rise to structural modifications of both polar and nonpolar lipid bilayer regions, viz. enhancement of membrane hydration and increase in tightness of lipid chain packing. The fact that EC accumulating in lipid bilayer are incapable of inducing significant disruption of membrane structural integrity creates strong prerequisites for development of liposomal nanocarriers of these potential antitumor drugs. Such a possibility is also corroborated by the observation that EC membrane incorporation does not prevent lipid bilayer partitioning of long-wavelength squaraine dyes which represent promising candidates for visualization of liposome biodistribution.