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Curcumin, a natural product with recognized antiviral properties, is limited in its application largely due to its poor solubility. This study presents the synthesis of water-soluble curcumin-poly(sodium 4-styrenesulfonate) (Cur-PSSNan) covalent conjugates. The antiflaviviral activity of conjugates was validated in vitro by using the Zika virus as a model. In the development of these water-soluble curcumin-containing derivatives, we used the macromolecules reported by us to also hamper viral infections. Mechanistic investigations indicated that the conjugates exhibited excellent stability and bioavailability. The curcumin and macromolecules in concerted action interact directly with virus particles and block their attachment to host cells, hampering the infection process.

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ß-Lactoglobulin (BLG) is a member of the lipocalin family. As other proteins from this group, BLG can be modified to bind specifically compounds of medical interests. The aim of this study was to evaluate the role of two mutations, L39Y and L58F, in the binding of topical anesthetic pramoxine (PRM) to ß-lactoglobulin. Circular dichroism spectroscopy, isothermal titration calorimetry (ITC), and X-ray crystallography were used to understand the mechanisms of BLG-PRM interactions. Studies were performed for three new BLG mutants: L39Y, L58F, and L39Y/L58F. ITC measurements indicated a significant increase in the affinity to the PRM of variants L58F and L39Y. Measurements taken for the double mutant L39Y/L58F showed the additivity of two mutations leading to about 80-fold increase in the affinity to PRM in comparison to natural protein BLG from bovine milk. The determined crystal structures revealed that pramoxine is accommodated in the ß-barrel interior of BLG mutants and stabilized by hydrophobic interactions. The observed additive effect of two mutations on drug binding opens the possibility for further designing of new BLG variants with high affinity to selected drugs.

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Among potential macromolecule-based pharmaceuticals, polycations seem particularly interesting due to their proven antimicrobial properties and use as vectors in gene therapy. This makes an understanding of the mechanisms of these molecules' interaction with living structures important, so the goal of this paper was to propose and carry out experiments that will allow us to characterize these phenomena. Of particular importance is the question of toxicity of such structures to mammalian cells and, in the work presented here, two lines, normal fibroblasts 3T3-L1 and A549 lung cancer, were used to determine this. In this work, three well-defined cationic derivatives of barley-derived betaglucans obtained in a reaction with glycidyltrimethylammonium chloride (BBGGTMAC) with different degrees of cationization (50, 70, and 100% per one glucose unit) and electrostatic charge were studied. The studies address interactions of these polymers with proteins (bovine serum proteins and BSA), nucleic acids (DNA), glycosaminoglycans (heparin), and biological membranes. The results described in this study make it possible to indicate that toxicity is most strongly influenced by interactions with biological membranes and is closely related to the electrostatic charge of the macromolecule. The presentation of this observation was the goal of this publication. This paper also shows, using fluorescently labeled variants of polymers, the penetration and impact on cell structure (only for the polymer with the highest substitution binding to cell membranes is observed) by using confocal and SEM (for the polymer with the highest degree of substitution, and the appearance of additional structures on the surface of the cell membrane is observed). The labeled polymers are also tools used together with dynamic light scattering and calorimetric titration to study their interaction with other biopolymers. As for the interactions with biological membranes, lipid Langmuir monolayers as model membrane systems were used.

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We show by extensive experimental characterization combined with molecular simulations that pH has a major impact on the assembly mechanism and properties of poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) complexes. A combination of dynamic light scattering (DLS) and laser Doppler velocimetry (LDV) is used to assess the complexation, charge state, and other physical characteristics of the complexes, isothermal titration calorimetry (ITC) is used to examine the complexation thermodynamics, and circular dichroism (CD) is used to extract the polypeptides' secondary structure. For enhanced analysis and interpretation of the data, analytical ultracentrifugation (AUC) is used to define the precise molecular weights and solution association of the peptides. Molecular dynamics simulations reveal the associated intra- and intermolecular binding changes in terms of intrinsic vs. extrinsic charge compensation, the role of hydrogen bonding, and secondary structure changes, aiding in the interpretation of the experimental data. We combine the data to reveal the pH dependency of PLL/PGA complexation and the associated molecular level mechanisms. This work shows that not only pH provides a means to control complex formation but also that the associated changes in the secondary structure and binding conformation can be systematically used to control materials assembly. This gives access to rational design of peptide materials via pH control.

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A series of poly(ethylene glycol)-block-poly(sodium 4-styrenesulfonate) (PEG-b-PSSNa) copolymers were synthesized, and their antiviral activity against Zika virus (ZIKV) was determined. The polymers inhibit ZIKV replication in vitro in mammalian cells at nontoxic concentrations. The mechanistic analysis revealed that the PEG-b-PSSNa copolymers interact directly with viral particles in a zipper-like mechanism, hindering their interaction with the permissive cell. The antiviral activity of the copolymers is well-correlated with the length of the PSSNa block, indicating that the copolymers' ionic blocks are biologically active. The blocks of PEG present in copolymers studied do not hinder that interaction. Considering the practical application of PEG-b-PSSNa and the electrostatic nature of the inhibition, the interaction between the copolymers and human serum albumin (HSA) was evaluated. The formation of PEG-b-PSSNa-HSA complexes in the form of negatively charged nanoparticles well-dispersed in buffer solution was observed. That observation is promising, given the possible practical application of the copolymers.

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Affinity chromatography (AC) is one of the techniques widely used for the purification of recombinant proteins. In our previous study, we presented a successful application of the Argi system [1] for the purification of recombinant proteins, based on the specific interaction between an arginine tag and a DNA aptamer. Exploring the possible application of positively charged peptide tags in the purification of recombinant proteins, in this study we developed and characterized an AC system based on the specific and reversible interaction between a DNA aptamer and a lysine tag (Lys-tag) comprising five lysine residues (5 K). We optimized the length of both the selected DNA aptamer and Lys-tag which were named B5K aptamer and 5K-tag, respectively. The results showed that the stability of the B5K aptamer and 5K-tag was dependent on the presence of potassium ions. The conditions for mild elution of 5K-tagged protein from B5K aptamer were determined. Our study proved that the developed system can be used for the purification of recombinant proteins from Escherichia coli total protein extracts.

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Unfractionated heparin (UFH) and enoxaparin (Enox) were substituted with a photoswitch (PS) showing quantitative trans-cis and cis-trans photoisomerizations. Long half-life of the cis photoisomer enabled comparison of the properties of heparins substituted with both PS photoisomers. Hydrodynamic diameter, Dh, of UFH-PS decreased upon trans-cis photoisomerization, the change being more pronounced for UFH-PS with a higher degree of substitution (DS), while Dh of Enox-PS did not significantly change. The anticoagulative properties of substituted heparins were significantly attenuated compared to non-substituted compounds. The interaction of UFH-PS with HSA, lysozyme, and protamine was studied with ITC. Under serum-free conditions, UFH-PS-trans with a high DS stimulated proliferation of murine fibroblasts, while UFH-PS-cis decreased the viability of these cells. Under serum conditions, both UFH-PS-cis and UFH-PS-trans decreased cell viability, the reduction for UFH-PS-cis being higher than that for UFH-PS-trans. Neither Enox-PS-trans nor Enox-PS-cis influenced the viability at concentrations prolonging aPTT, while at higher concentrations their cytotoxicity did not differ.

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Human protein Yin Yang 1 (YY1) controls the transcription of hundreds of genes both positively and negatively through interactions with a wide range of partner proteins. Results presented here from proteolytic sensitivity, calorimetry, circular dichroism, fluorescence, NMR, size-exclusion chromatography, SELEX, and EMSA show that purified YY1 forms dimers via its disordered N-terminal region with strong zinc-ion concentration dependence. The YY1 dimer is shown to bind tandem repeats of a canonical recognition DNA sequence with high affinity, and analysis of human YY1 regulatory sites shows that many contain repeats of its recognition elements. YY1 dimerization may compete with partner protein interactions, making control by zinc ion concentration a previously unrecognized factor affecting YY1 gene regulation. Indeed, YY1 is known to be important in many pathogenic processes, including neoplasia, in which zinc ion concentrations are altered. The present results incentivize studies in vivo or in vitro that explore the role of zinc ion concentration in YY1-mediated gene expression.

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This work reports the results of random mutagenesis of the Escherichia coli class 2 L-asparaginase EcAIII belonging to the Ntn-hydrolase family. New variants of EcAIII were studied using structural, biophysical and bioinformatic methods. Activity tests revealed that the L-asparaginase activity is abolished in all analyzed mutants with the absence of Arg207, but some of them retained the ability to undergo the autoproteolytic maturation process. The results of spectroscopic studies and the determined crystal structures showed that the EcAIII fold is flexible enough to accept different types of mutations; however, these mutations may have a diverse impact on the thermal stability of the protein. The conclusions from the experiments are grouped into six lessons focused on (i) the adaptation of the EcAIII fold to new substitutions, (ii) the role of Arg207 in EcAIII activity, (iii) a network of residues necessary for autoprocessing, (iv) the complexity of the autoprocessing reaction, (v) the conformational changes observed in enzymatically inactive variants and (vi) the cooperativity of the EcAIII dimer subunits. Additionally, the structural requirements (pre-maturation checkpoints) that are necessary for the initiation of the autocleavage of Ntn-hydrolases have been classified. The findings reported in this work provide useful hints that should be considered before planning enzyme-engineering experiments aimed at the design of proteins for therapeutic applications. This is especially important for L-asparaginases that can be utilized in leukemia therapy, as alternative therapeutics are urgently needed to circumvent the severe side effects associated with the currently used enzymes.

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The homodimeric ß-lactoglobulin belongs to the lipocalin family of proteins that transport a wide range of hydrophobic molecules and can be modified by mutagenesis to develop specificity for novel groups of ligands. In this work, new lactoglobulin variants, FAF (I56F/L39A/M107F) and FAW (I56F/L39A/M107W), were produced and their interactions with the tricyclic drug desipramine (DSM) were studied using X-ray crystallography, calorimetry (ITC) and circular dichroism (CD). The ITC and CD data showed micromolar affinity of the mutants for DSM and interactions according to the classical one-site binding model. However, the crystal structures unambiguously showed that the FAF and FAW dimers are capable of binding DSM not only inside the ß-barrel as expected, but also at the dimer interface and at the entrance to the binding pocket. The presented high-resolution crystal structures therefore provide important evidence of the existence of alternative ligand-binding sites in the ß-lactoglobulin molecule. Analysis of the crystal structures highlighted the importance of shape complementarity for ligand recognition and selectivity. The binding sites identified in the crystal structures of the FAF-DSM and FAW-DSM complexes together with data from the existing literature are used to establish a systematic classification of the ligand-binding sites in the ß-lactoglobulin molecule.

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Physicochemical properties of poly-L-arginine (P-Arg) molecules in NaCl solutions were determined by molecular dynamics (MD) modeling and various experimental techniques. Primarily, the molecule conformations, the monomer length and the chain diameter were theoretically calculated. These results were used to interpret experimental data, which comprised the molecule secondary structure, the diffusion coefficient, the hydrodynamic diameter and the electrophoretic mobility determined at various ionic strengths and pHs. Using these data, the electrokinetic charge and the effective ionization degree of P-Arg molecules were determined. In addition, the dynamic viscosity measurements for dilute P-Arg solutions enabledto determine the molecule intrinsic viscosity, which was equal to 500 and 90 for ionic strength of 10-5 and 0.15 M, respectively. This confirmed that P-Arg molecules assumed extended conformations and approached the slender body limit at the low range of ionic strength. The experimental data were also used to determine the molecule length and the chain diameter, which agreed with theoretical predictions. Exploiting these results, a robust method for determining the molar mass of P-Arg samples, the hydrodynamic diameter, the radius of gyration and the sedimentation coefficient was proposed.

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ß-Lactoglobulin (BLG), a member of the lipocalin family, is a well-studied model protein. It is also widely used as a scaffold for the development of novel proteins. Our previous work adopted a rational approach based on homolog structure alignment to obtain several BLG variants with point mutations inside the binding pocket. To investigate the effect of mutation on ligand binding thermodynamics, we chose a set of aliphatic ligands and performed a study based on isothermal titration calorimetry. In addition, the circular dichroism spectra observed for the protein-ligand complexes were analyzed. The ligand binding thermodynamics was compared between wild-type and mutated BLG as well as between two ligands. The findings pointed to factors that can be responsible for the mutation-induced changes in the thermodynamics of the complexes.

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The potential-sensitive di-4-ANEPPDHQ dye is presently gaining popularity in structural studies of the lipid bilayer. Within the bilayer, dye environmental sensitivity originates from the excitation induced charge redistribution and is usually attributed to solvent relaxation. Here, di-4-ANEPPDHQ is utilized to compare the structure of neutral and negatively charged lipid bilayers between two model systems: the nanodiscs and the liposomes. Using the well-established approach of measuring solvatochromic shifts of the steady-state spectra to study the bilayer structural changes has proved insufficient in this case. By applying an in-depth analysis of time-resolved fluorescence decays and emission spectra, we distinguished and characterized two and three distinct emissive di-4-ANEPPDHQ species in the liposomes and the nanodiscs, respectively. These emissive species were ascribed to the dual emission of the dye rather than to solvent relaxation. An additional, long-lived component present in the nanodiscs was associated with a unique domain of high order, postulated recently. Our results reveal that the di-4-ANEPPDHQ steady-state fluorescence should be interpreted with caution. With the experimental approach presented here, the di-4-ANEPPDHQ sensitivity was improved. We confirmed that the bilayer structure is, indeed, altered in the nanodiscs. Moreover, molecular dynamic simulations showed a distribution of the probe in the nanodiscs plane, which is sensitive to lipid composition. In POPC nanodiscs, probe frequently interacts with MSP, while in POPC-POPG nanodiscs, such interactions are rare. We did not observe, however, any impact of those interactions on the probe fluorescence.

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ß-Lactoglobulin (BLG) like other lipocalins can be modified by mutagenesis to re-direct its ligand binding properties. Local site-directed mutagenesis was used to change the geometry of the BLG ligand binding pocket and therefore change BLG ligand preferences. The presented studies are focused on previously described mutants L39Y, I56F, L58F, F105L, and M107L and two new BLG variants, L39K and F105A, and their interactions with local anesthetic drug tetracaine. Binding of tetracaine to BLG mutants was investigated by X-ray crystallography. Structural analysis revealed that for tetracaine binding, the shape of the binding pocket seems to be a more important factor than the substitutions influencing the number of interactions. Analyzed BLG mutants can be classified according to their binding properties to variants: capable of binding tetracaine in the ß-barrel (L58F, M107L); capable of accommodating tetracaine on the protein surface (I56F) and unable to bind tetracaine (F105L). Variants L39K, L39Y, and F105A, had a binding pocket blocked by endogenous fatty acids. The new tetracaine binding site was found in the I56F variant. The site localized on the surface near Arg124 and Trp19 was previously predicted by in silico studies and was confirmed in the crystal structure.

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Microsecond-long all-atom molecular dynamics (MD) simulations, circular dichroism, laser Doppler velocimetry, and dynamic light-scattering techniques have been used to investigate pH-induced changes in the secondary structure, charge, and conformation of poly l-lysine (PLL) and poly l-glutamic acid (PGA). The employed combination of the experimental methods reveals for both PLL and PGA a narrow pH range at which they are charged enough to form stable colloidal suspensions, maintaining their α-helix content above 60%; an elevated charge state of the peptides required for colloidal stability promotes the peptide solvation as a random coil. To obtain a more microscopic view on the conformations and to verify the modeling performance, peptide secondary structure and conformations rising in MD simulations are also examined using three different force fields, i.e., OPLS-AA, CHARMM27, and AMBER99SB*-ILDNP. Ramachandran plots reveal that in the examined setup the α-helix content is systematically overestimated in CHARMM27, while OPLS-AA overestimates the ß-sheet fraction at lower ionization degrees. At high ionization degrees, the OPLS-AA force-field-predicted secondary structure fractions match the experimentally measured distribution most closely. However, the pH-induced changes in PLL and PGA secondary structure are reasonably captured only by the AMBER99SB*-ILDNP force field, with the exception of the fully charged PGA in which the α-helix content is overestimated. The comparison to simulations results shows that the examined force fields involve significant deviations in their predictions for charged homopolypeptides. The detailed mapping of secondary structure dependency on pH for the polypeptides, especially finding the stable colloidal α-helical regime for both examined peptides, has significant potential for practical applications of the charged homopolypeptides. The findings raise attention especially to the pH fine tuning as an underappreciated control factor in surface modification and self-assembly.

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Recombinant proteins play an important role in medicine and have diverse applications in industrial biotechnology. Lactoglobulin has shown great potential for use in targeted drug delivery and body fluid detoxification because of its ability to bind a variety of molecules. In order to modify the biophysical properties of ß-lactoglobulin, a series of single-site mutations were designed using a structure-based approach. A 3-dimensional structure alignment of homologous molecules led to the design of nine ß-lactoglobulin variants with mutations introduced in the binding pocket region. Seven stable and correctly folded variants (L39Y, I56F, L58F, V92F, V92Y, F105L, M107L) were thoroughly characterized by fluorescence, circular dichroism, isothermal titration calorimetry, size-exclusion chromatography, and X-ray structural investigations. The effects of the amino acid substitutions were observed as slight rearrangements of the binding pocket geometry, but they also significantly influenced the global properties of the protein. Most of the mutations increased the thermal/chemical stability without altering the dimerization constant or pH-dependent conformational behavior. The crystal structures reveal that the I56F and F105L mutations reduced the depth of the binding pocket, which is advantageous since it can reduce the affinity to endogenous fatty acids. The F105L mutant created a unique binding mode for a fatty acid, supporting the idea that lactoglobulin can be altered to bind unique molecules. Selected variants possessing a unique combination of their individual properties can be used for further, more advanced mutagenesis, and the presented results support further research using ß-lactoglobulin as a therapeutic delivery agent or a blood detoxifying molecule.

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Ovine ß­lactoglobulin was characterized by spectroscopic (CD), calorimetric (ITC) and X-ray structural studies. The structure of ovine ß­lactoglobulin complex with decanol showed that tight packing of molecules in the crystalline phase enforces a distortion of protein flexible loops resulting in the formation of an asymmetric dimer. The loops surrounding ß-barrel in ovine lactoglobulin possessed the same conformational flexibility as observed previously in other lactoglobulins and the change of their conformation regulates the access to the binding pocket. The structure of asymmetric dimer revealed a new region in ß-barrel where ligand polar group can be located. These findings indicated protein adaptability to ligand dimensions and inter- and intramolecular interactions in the crystalline phase. Calorimetric and crystallographic studies provided the experimental evidence that ovine lactoglobulin is able to bind aliphatic ligands. Thermodynamic parameters of sodium dodecyl sulfate binding determined by ITC at pH 7.5 had Ka, ΔH, TΔS and ΔG values similar to those observed for bovine and caprine protein indicating the same mechanism of ligand binding.

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In proteins, proton exchange is caused due to the changes in the proton affinity (pKa) of ionizable groups that are engaged in conformational changes induced by the binding of a ligand. In addition, knowledge regarding the type and number of such ionizable groups is very important to understand the pH-dependent changes of the thermodynamic parameters. Therefore, in this study, we performed a comprehensive analysis of proton exchange by using beta lactoglobulin (Blg), a representative of the lipocalin family of proteins. We used isothermal titration calorimetry to evaluate the proton exchange during binding with sodium dodecyl sulfate (SDS) at different pHs ranging from 2 to 9. SDS binds to Blg in all studied range of pHs and enthalpy-driven reactions are observed in acidic pH, whereas enthalpy-entropy driven reactions are observed in neutral and basic pHs. Enthalpy-entropy compensation leads to relatively small changes in the association constants with the maximal value at pH = 8.0. The simultaneous analysis of the number of exchanged protons, the binding constants, and the enthalpy was performed in the range pH 5.5-9. At least 4 residues that change their ionization pattern are needed to explain the observed pH dependence.

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Chlorpromazine (CPZ) is a phenothiazine acting as dopamine antagonist. Aside from application in schizophrenia therapy, chlorpromazine is found to be a putative inhibitor of proteins involved in cancers, heritable autism disorder and prion diseases. Four new ß-lactoglobulin variants with double or triple substitutions: I56F/L39A, F105L/L39A, I56F/L39A/M107F or F105L/L39A/M107F changing the shape of the binding pocket were produced and their chlorpromazine binding properties have been investigated by X-ray crystallography, circular dichroism, isothermal titration calorimetry and thermophoresis. The CD spectra and crystal structures revealed that mutations do not affect the protein overall structure but in comparison to WT protein, variants possessing I56F substitution had lower stability while mutation F105L increased melting temperature of the protein. The new variants showed affinity to chlorpromazine in the range 4.2-15.4 × 103 M-1. The CD spectra and crystal structures revealed complementarity of the binding pocket shape, to only one chlorpromazine chiral conformer. The (aR)-CPZ was bonded to variants containing I56F substitution while variants with F105L substitution preferred (aS)-CPZ.

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The TNF-Related Apoptosis Inducing Ligand (TRAIL) cytokine triggers apoptosis specifically in cancer cells. Susceptibility of a given cell to TRAIL depends on the activity of regulatory proteins, one of the most important of which is BID. The aim of this study was to increase the cytotoxic potential of TRAIL against cancer cells. TRAIL was fused to the BH3 domain of BID. Hence, TRAIL acted not only as an anticancer agent, but also as a specific carrier for the BID fragment. Two fusion protein variants were obtained by genetic engineering, harboring two different linker sequences. The short linker allowed both parts of the fusion protein to fold into their native structures. The long linker influenced the structure of the fused proteins but nonetheless resulted in their highest cytotoxic activity. Optimal buffer formulation was determined for all the analyzed TRAIL variants. Fusing the BH3 domain of BID to TRAIL improved the cytotoxic potential of TRAIL. Further, these findings may be useful for the optimization of other anticancer drugs based on TRAIL, since the appropriate formulation would secure their native structures during prolonged storage.

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