Charge Regulation Triggers Condensation of Short Oligopeptides to Polyelectrolytes.

Electrostatic interactions between charged macromolecules are ubiquitous in biological systems, and they are important also in materials design. Attraction between oppositely charged molecules is often interpreted as if the molecules had a fixed charge, which is not affected by their interaction. Less commonly, charge regulation is invoked to interpret such interactions, i.e., a change of the charge state in response to a change of the local environment. Although some theoretical and simulation studies suggest that charge regulation plays an important role in intermolecular interactions, experimental evidence supporting such a view is very scarce. In the current study, we used a model system, composed of a long polyanion interacting with cationic oligolysines, containing up to 8 lysine residues. We showed using both simulations and experiments that while these lysines are only weakly charged in the absence of the polyanion, they charge up and condense on the polycations if the pH is close to the pKa of the lysine side chains. We show that the lysines coexist in two distinct populations within the same solution: (1) practically nonionized and free in solution; (2) highly ionized and condensed on the polyanion. Using this model system, we demonstrate under what conditions charge regulation plays a significant role in the interactions of oppositely charged macromolecules and generalize our findings beyond the specific system used here.

Stimuli-Responsive Triblock Terpolymer Conversion into Multi-Stimuli-Responsive Micelles with Dynamic Covalent Bonds for Drug Delivery through a Quick and Controllable Post-Polymerization Reaction.

Stimuli-responsive copolymers are of great interest for targeted drug delivery. This study reports on a controllable post-polymerization quaternization with 2-bromomethyl-4-fluorophenylboronic acid of the poly(4-vinyl pyridine) (P4VP) block of a common poly(styrene)-b-poly(4-vinyl pyridine)-b-poly(ethylene oxide) (SVE) triblock terpolymer in order to achieve a selective responsivity to various diols. For this purpose, a reproducible method was established for P4VP block quaternization at a defined ratio, confirming the reaction yield by 11B, 1H NMR. Then, a reproducible self-assembly protocol is designed for preparing stable micelles from functionalized stimuli-responsive triblock terpolymers, which are characterized by light scattering and by cryogenic transmission electron microscopy. In addition, UV-Vis spectroscopy is used to monitor the boron-ester bonding and hydrolysis with alizarin as a model drug and to study encapsulation and release of this drug, induced by sensing with three geminal diols: fructose, galactose and ascorbic acid. The obtained results show that only the latter, with the vicinal diol group on sp2-hybridized carbons, was efficient for alizarin release. Therefore, the post-polymerization method for triblock terpolymer functionalization presented in this study allows for preparation of specific stimuli-responsive systems with a high potential for targeted drug delivery, especially for cancer treatment.

Anionically Functionalized Glycogen Encapsulates Melittin by Multivalent Interaction.

We developed acid-functionalized glycogen conjugates as supramolecular carriers for efficient encapsulation and inhibition of a model cationic peptide melittin─the main component of honeybee venom. For this purpose, we synthesized and characterized a set of glycogens, functionalized to various degrees by several different acid groups. These conjugates encapsulate melittin up to a certain threshold amount, beyond which they precipitate. Computer simulations showed that sufficiently functionalized conjugates electrostatically attract melittin, resulting in its efficient encapsulation in a broad pH range around the physiological pH. Hemolytic assays confirmed in vitro that the effective inhibition of melittin's hemolytic activity occurs for highly functionalized samples, whereas no inhibition is observed when using low-functionalized conjugates. It can be concluded that functional glycogens are promising carriers for cationic molecular cargos or antidotes against animal venoms under conditions, in which suitable properties such as biodegradability and biocompatibility are crucial.

DPD Modelling of the Self- and Co-Assembly of Polymers and Polyelectrolytes in Aqueous Media: Impact on Polymer Science.

This review article is addressed to a broad community of polymer scientists. We outline and analyse the fundamentals of the dissipative particle dynamics (DPD) simulation method from the point of view of polymer physics and review the articles on polymer systems published in approximately the last two decades, focusing on their impact on macromolecular science. Special attention is devoted to polymer and polyelectrolyte self- and co-assembly and self-organisation and to the problems connected with the implementation of explicit electrostatics in DPD numerical machinery. Critical analysis of the results of a number of successful DPD studies of complex polymer systems published recently documents the importance and suitability of this coarse-grained method for studying polymer systems.

Reversible multilayered vesicle-like structures with fluid hydrophobic and interpolyelectrolyte layers.

Hydrophobic blocks of amphiphilic block copolymers often form glassy micellar cores at room temperature with a rigid structure that limits their applications as nanocapsules for targeted delivery. Nevertheless, we prepared and analyzed core/shell micelles with a soft core, formed by a self-assembled block copolymer consisting of a hydrophobic block and a polycation block, poly(lauryl acrylate)-block-poly(trimethyl-aminoethyl acrylate) (PLA-QPDMAEA), in aqueous solution. By light and small-angle neutron scattering, by transmission electron microscopy and by fluorescence spectroscopy, we showed that these core/shell micelles are spherical and cylindrical with a fluid-like PLA core and a positively charged outer shell and that they can encapsulate and release hydrophobic solutes. Moreover, after mixing these PLA-QPDMAEA core/shell micelles with another diblock copolymer, consisting of a hydrophilic block and a polyanion block, namely poly(ethylene oxide)-block-poly(methacrylic acid) (PEO-PMAA), we observed the formation of novel vesicle-like multicompartment structures containing both soft hydrophobic and interpolyelectrolyte (IPEC) layers. By combining small-angle neutron scattering with self-consistent field modeling, we confirmed the formation of these complex vesicle-like structures with a swollen PEO core, an IPEC inner layer, a PLA soft layer, an IPEC outer layer and a loose PEO corona. Thus, these multicompartment micelles with fluid and IPEC layers and a hydrophilic corona may be used as nanocapsules with several tunable properties, including the ability to control the thickness of each layer, the charge of the IPEC layers and the stability of the micelles, to deliver both hydrophobic and multivalent solutes.

Role of pKA in Charge Regulation and Conformation of Various Peptide Sequences.

Peptides containing amino acids with ionisable side chains represent a typical example of weak ampholytes, that is, molecules with multiple titratable acid and base groups, which generally exhibit charge regulating properties upon changes in pH. Charged groups on an ampholyte interact electrostatically with each other, and their interaction is coupled to conformation of the (macro)molecule, resulting in a complex feedback loop. Their charge-regulating properties are primarily determined by the pKA of individual ionisable side-chains, modulated by electrostatic interactions between the charged groups. The latter is determined by the amino acid sequence in the peptide chain. In our previous work we introduced a simple coarse-grained model of a flexible peptide. We validated it against experiments, demonstrating its ability to quantitatively predict charge on various peptides in a broad range of pH. In the current work, we investigated two types of peptide sequences: diblock and alternating, each of them consisting of an equal number of amino acids with acid and base side-chains. We showed that changing the sequence while keeping the same overall composition has a profound effect on the conformation, whereas it practically does not affect total charge on the peptide. Nevertheless, the sequence significantly affects the charge state of individual groups, showing that the zero net effect on the total charge is a consequence of unexpected cancellation of effects. Furthermore, we investigated how the difference between the pKA of acid and base side chains affects the charge and conformation of the peptide, showing that it is possible to tune the charge-regulating properties by following simple guiding principles based on the pKA and on the amino acid sequence. Our current results provide a theoretical basis for understanding of the complex coupling between the ionisation and conformation in flexible polyampholytes, including synthetic polymers, biomimetic materials and biological molecules, such as intrinsically disordered proteins, whose function can be regulated by changes in the pH.

Physicochemical Evaluation of Insulin Complexes with QPDMAEMA-b-PLMA-b-POEGMA Cationic Amphiphlic Triblock Terpolymer Micelles.

Herein, poly[quaternized 2-(dimethylamino)ethyl methacrylate-b-lauryl methacrylate-b-(oligo ethylene glycol)methacrylate] (QPDMAEMA-b-PLMA-b-POEGMA) cationic amphiphilic triblock terpolymers were used as vehicles for the complexation/encapsulation of insulin (INS). The terpolymers self-assemble in spherical micelles with PLMA cores and mixed QPDMAEMA/POEGMA coronas in aqueous solutions. The cationic micelles were complexed via electrostatic interactions with INS, which contains anionic charges at pH 7. The solutions were colloidally stable in all INS ratios used. Light-scattering techniques were used for investigation of the complexation ability and the size and surface charge of the terpolymer/INS complexes. The results showed that the size of the complexes increases as INS ratio increases, while at the same time the surface charge remains positive, indicating the formation of clusters of micelles/INS complexes in the solution. Fluorescence spectroscopy measurements revealed that the conformation of the protein is not affected after the complexation with the terpolymer micellar aggregates. It was observed that as the solution ionic strength increases, the size of the QPDMAEMA-b-PLMA-b-POEGMA/INS complexes initially decreases and then remains practically constant at higher ionic strength, indicating further aggregation of the complexes. atomic force microscopy (AFM) measurements showed the existence of both clusters and isolated nanoparticulate terpolymer/protein complexes.

Polystyrene and Poly(ethylene glycol)-b-Poly(ε-caprolactone) Nanoparticles with Porphyrins: Structure, Size, and Photooxidation Properties.

The transport of a photosensitizer to target biological structures followed by the release of singlet oxygen is a critical step in photodynamic therapy. We compared the (photo)physical properties of polystyrene nanoparticles (TPP@PS) of different sizes and self-assembled poly(ethylene glycol)-b-poly(ε-caprolactone) core/shell nanoparticles (TPP@PEG-PCL) with different lengths of copolymer blocks, both suitable for the transport of the tetraphenylporphyrin (TPP) photosensitizer. The singlet oxygen was formed inside both nanoparticles after irradiation with visible light. Its kinetics was controlled by the size of TPP@PS; its lifetime (τΔ) increased with increasing nanoparticle size (from 6.5 to 16 µs) because of hindered diffusion into the external aqueous environment, where it was quickly deactivated. Accordingly, the prolongation of the singlet oxygen-sensitized delayed fluorescence kinetics was found for TPP@PS of high size. The TPP@PEG-PCL self-assemblies allowed for enhanced oxygen diffusion, and the estimated low values of τΔ ≈ 3.7 µs were independent of the size of building blocks. The delayed fluorescence in oxygen-free conditions originating from triplet-triplet annihilation indicated a high mobility of TPP in the PCL core in comparison with fixed molecules in the PS matrix. Photooxidation of uric acid revealed the highest efficacy for TPP@PS of small sizes, whereas the largest TPP@PS exhibited the lowest activity, and the efficacy of TPP@PEG-PCL remained independent of the sizes of the building blocks.

Formation of core/corona nanoparticles with interpolyelectrolyte complex cores in aqueous solution: insight into chain dynamics in the complex from fluorescence quenching.

Formation of interpolyelectrolyte complexes (IPECs) of poly(methacrylic acid) (PMAA) bearing a fluorescent label (umbelliferone) at the chain end and poly[3,5-bis(trimethyl ammoniummethyl)-4-hydroxystyrene iodide]-block-poly(ethylene oxide) (QNPHOS-PEO) acting as a fluorescence quencher, was followed using a combination of scattering, calorimetry, microscopy and fluorescence spectroscopy techniques. While scattering and microscopy measurements indicated formation of spherical core/corona nanoparticles with the core of the QNPHOS/PMAA complex and the PEO corona, fluorescence measurements showed that both static and dynamic quenching efficiency were increased in the nanoparticle stability region. As the dynamic quenching rate constant remained unchanged, the quenching enhancement was caused by the increase in the local concentration of QNPHOS segments in the microenvironment of the label. This finding implies that the local dynamics of PMAA end chains affecting the interaction of the label with QNPHOS segments was independent of both PMAA and QNPHOS chain conformations.

Local pH and Effective pK of a Polyelectrolyte Chain: Two Names for One Quantity?

In recent experiments, the "local pH" near polyelectrolyte chains was determined from the shift in the effective acidity constant of fluorescent pH indicators attached to the macromolecules. This indirect determination raises the question if the analyzed quantity was indeed the "local pH" and what this term actually means. In this study, we combined experiments and simulations to demonstrate that the shift in ionization constant is slightly lower than the difference between the pH and the "local pH". This offset is caused by correlations between fluctuations in chain conformation, small-ion distribution, and fluorophore ionization.

Nanoparticles with Embedded Porphyrin Photosensitizers for Photooxidation Reactions and Continuous Oxygen Sensing.

We report the synthesis and characterization of sulfonated polystyrene nanoparticles (average diameter 30 ± 14 nm) with encapsulated 5,10,15,20-tetraphenylporphyrin or ionically entangled tetracationic 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin, their photooxidation properties, and the application of singlet oxygen-sensitized delayed fluorescence (SODF) in oxygen sensing. Both types of nanoparticles effectively photogenerated singlet oxygen, O2(1Δg). The O2(1Δg) phosphorescence, transient absorption of the porphyrin triplet states, and SODF signals were monitored using time-resolved spectroscopic techniques. The SODF intensity depended on the concentration of the porphyrin photosensitizer and dissolved oxygen and on the temperature. After an initial period (a few microseconds), the kinetics of the SODF process can be approximated as a monoexponential function, and the apparent SODF lifetimes can be correlated with the oxygen concentration. The oxygen sensing based on SODF allowed measurement of the dissolved oxygen in aqueous media in the broad range of oxygen concentrations (0.2-38 mg L-1). The ability of both types of nanoparticles to photooxidize external substrates was predicted by the SODF measurements and proven by chemical tests. The relative photooxidation efficacy was highest at a low porphyrin concentration, as indicated by the highest fluorescence quantum yield (ΦF), and it corresponds with negligible inner filter and self-quenching effects. The photooxidation abilities were sensitive to the influence of temperature on the diffusion and solubility of oxygen in both polystyrene and water media and to the rate constant of the O2(1Δg) reaction with a substrate. Due to their efficient photogeneration of cytotoxic O2(1Δg) at physiological temperatures and their oxygen sensing via SODF, both types of nanoparticles are promising candidates for biomedical applications.

Composite particles formed by complexation of poly(methacrylic acid) - stabilized magnetic fluid with chitosan: Magnetic material for bioapplications.

A simple procedure for the synthesis of magnetic fluid (ferrofluid) stabilized by poly(methacrylic acid) has been developed. This ferrofluid was used to prepare a novel type of magnetically responsive chitosan-based composite material. Both ferrofluid and magnetic chitosan composite were characterized by a combination of microscopy (optical microscopy, TEM, SEM), scattering (static and dynamic light scattering, SANS) and spectroscopy (FTIR) techniques. Magnetic chitosan was found to be a perspective material for various bioapplications, especially as a magnetic carrier for immobilization of enzymes and cells. Lipase from Candida rugosa was covalently attached after cross-linking and activation of chitosan using glutaraldehyde. Baker's yeast cells (Saccharomyces cerevisiae) were incorporated into the chitosan composite during its preparation; both biocatalysts were active after reaction with appropriate substrates.

Influence of Corona Structure on Binding of an Ionic Surfactant in Oppositely Charged Amphiphilic Polyelectrolyte Micelles.

Interaction of polystyrene-block-poly(methacrylic acid) micelles (PS-PMAA) with cationic surfactant N-dodecylpyridinium chloride (DPCl) in alkaline aqueous solutions was studied by static and dynamic light scattering, SAXS, cryogenic transmission electron microscopy (cryo-TEM), isothermal titration calorimetry (ITC), and time-resolved fluorescence spectroscopy. ITC and fluorescence measurements show that there are two distinct regimes of surfactant binding in the micellar corona (depending on the DPCl content) caused by different interactions of DPCl with PMAA in the inner and outer parts of the corona. The compensation of the negative charge of the micellar corona by DPCl leads to the aggregation of PS-PMAA micelles, and the micelles form colloidal aggregates at a certain critical surfactant concentration. SAXS shows that the aggregates are formed by individual PS-PMAA micelles with intact cores and collapsed coronas interconnected with surfactant micelles by electrostatic interactions. Unlike polyelectrolyte-surfactant complexes formed by free polyelectrolyte chains, the PMAA/DPCl complex with collapsed corona does not contain surfactant micelles.

Glucose-Responsive Hybrid Nanoassemblies in Aqueous Solutions: Ordered Phenylboronic Acid within Intermixed Poly(4-hydroxystyrene)-block-poly(ethylene oxide) Block Copolymer.

Coassembly behavior of the double hydrophilic block copolymer poly(4-hydroxystyrene)-block-poly(ethylene oxide) (PHOS-PEO) with three amphiphilic phenylboronic acids (PBA) differing in hydrophobicity, 4-dodecyloxyphenylboronic acid (C12), 4-octyloxyphenylboronic acid (C8), and 4-isobutoxyphenylboronic acid (i-Bu) was studied in alkaline aqueous solutions and in mixtures of NaOHaq/THF by spin-echo (1)H NMR spectroscopy, dynamic and electrophoretic light scattering, and SAXS. The study reveals that only the coassembly of C12 with PHOS-PEO provides spherical nanoparticles with intermixed PHOS and PEO blocks, containing densely packed C12 micelles. NMR measurements have shown that spatial proximity of PHOS-PEO and C12 leads to the formation of ester bonds between -OH of PHOS block and hydroxyl groups of -B(OH)2. Due to the presence of PBA moieties, the release of compounds with 1,2- or 1,3-dihydroxy groups loaded in the coassembled PHOS-PEO/PBA nanoparticles by covalent binding to PBA can be triggered by addition of a surplus of glucose that bind to PBA competitively. The latter feature has been confirmed by fluorescence measurements using Alizarin Red as a model compound. Nanoparticles were proved to exhibit swelling in response to glucose as detected by light scattering.

Steady-state and time-resolved luminescence of Ru(II) polypyridine complexes attached to Ag nanoparticles: Effect of chemisorption in comparison with electrostatic bonding.

Steady state and nanosecond time resolved luminescence (namely, (3)MLCT phosphorescence) of [Ru(bpy)3](2+) and of [Ru(bpy)2(dcbpy)](2+)/bpy=2,2'-bipyridine; dcbpy=2,2'-bipyridyl-4,4'-dicarboxylic acid/attached to Ag NPs (the former by the electrostatic bonding, the latter by chemisorption) in non-aggregated Ag NP hydrosol systems has been investigated, and compared to the luminescence characteristics of the complexes in aqueous solutions. The intensity decrease of the 452 nm (and/or 455 nm, respectively) main band and elimination of the short wavelength shoulders in the excitation spectra and the intensity decrease of the emission spectra observed for both complexes upon their attachment to Ag NPs is attributed to the overlap of the excitation spectra with the surface plasmon extinction (SPE) of Ag NPs. The overlap leads to a loss of excitation energy by SPE as well as to a decrease of the (1)MLCT to (3)MLCT intersystem crossing efficiency. The time-resolved luminescence study shows that the (3)MLCT phosphorescence lifetimes of both complexes are markedly (by 3 and 4 orders of magnitude, respectively) shortened upon their attachment to Ag NPs. Nevertheless, the (3)MLCT lifetime of the chemisorbed [Ru(bpy)2(dcbpy)](2+) is by at least one order of magnitude shorter than that of the electrostatically bonded [Ru(bpy)3](2+), which indicates, that the phosphorescence lifetimes of these luminophores are strongly affected by the type of Ag NP surface-luminophore bonding.

Polyelectrolyte-surfactant complexes of poly[3,5-bis(dimethylaminomethyl)-4-hydroxystyrene]-block-poly(ethylene oxide) and sodium dodecyl sulfate: anomalous self-assembly behavior.

Polyelectrolyte-surfactant complexes (PE-S) formed by double hydrophilic cationic polyelectrolyte poly[3,5-bis(dimethylaminomethyl)-4-hydroxystyrene]-block-poly(ethylene oxide) (NPHOS-PEO) and anionic surfactant sodium dodecyl sulfate (SDS) in acidic aqueous solutions were studied by light scattering, SAXS, and scanning transmission electron microcopy in the environmental mode (wet-STEM) for various stoichiometric ratios between the numbers of SDS anions and dimethylaminomethyl groups of NPHOS in the complex. The obtained results show that the NPHOS-PEO/SDS system behaves differently from other systems of double hydrophilic block polyelectrolyte and oppositely charged ionic surfactant because it forms water-insoluble PE-S for compositions close to the zero net charge of the complex. This phase separation occurs, instead of the PE-S rearrangement to core-shell particles, which is hindered due to conformational rigidity of the NPHOS blocks. For the surfactant amounts below and above the precipitation region, large spherical aggregates and their clusters are present in the solution. SAXS measurements indicate that although the NPHOS-PEO/SDS system does not form the core-shell particles with the NPHOS/SDS core and the PEO shell as other PE-S of double hydrophilic polyelectrolytes, the aggregates contain domains of closely packed surfactant micelles which bind to both NPHOS polyelectrolyte blocks and PEO blocks.

Structural modulation of phosducin by phosphorylation and 14-3-3 protein binding.

Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the G(t)ßγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function.

Association of poly(4-hydroxystyrene)-block-poly(ethylene oxide) in aqueous solutions: block copolymer nanoparticles with intermixed blocks.

Association behavior of diblock copolymer poly(4-hydroxystyrene)-block-poly(ethylene oxide) (PHOS-PEO) in aqueous solutions and solutions in water/tetrahydrofuran mixtures was studied by static, dynamic, and electrophoretic light scattering, (1)H NMR spectroscopy, transmission electron microscopy, and cryogenic field-emission scanning electron microscopy. It was found that, in alkaline aqueous solutions, PHOS-PEO can form compact spherical nanoparticles whose size depends on the preparation protocol. Instead of a core/shell structure with segregated blocks, the PHOS-PEO nanoparticles have intermixed PHOS and PEO blocks due to hydrogen bond interaction between -OH groups of PHOS and oxygen atoms of PEO and are stabilized electrostatically by a fraction of ionized PHOS units on the surface.

Polyelectrolyte-surfactant complexes formed by poly[3,5-bis(trimethylammoniummethyl)4-hydroxystyrene iodide]-block-poly(ethylene oxide) and sodium dodecyl sulfate in aqueous solutions.

Formation of polyelectrolyte-surfactant (PE-S) complexes of poly[3,5-bis(trimethylammoniummethyl)-4-hydroxystyrene iodide]-block-poly(ethylene oxide) (QNPHOS-PEO) and sodium dodecyl sulfate (SDS) in aqueous solution was studied by dynamic and electrophoretic light scattering, small-angle X-ray scattering (SAXS), atomic force microscopy, and fluorometry, using pyrene as a fluorescent probe. SAXS data from the QNPHOS-PEO/SDS solutions were fitted assuming contributions from free copolymer, PE-S aggregates described by a mass fractal model, and densely packed surfactant micelles inside the aggregates. It was found that, unlike other systems of a double hydrophilic block polyelectrolyte and an oppositely charged surfactant, PE-S aggregates of the QNPHOS-PEO/SDS system do not form core-shell particles and the PE-S complex precipitates before reaching the charge equivalence between dodecyl sulfate anions and QNPHOS polycationic blocks, most likely because of conformational rigidity of the QNPHOS blocks, which prevents the system from the corresponding rearrangement.

The C-terminal segment of yeast BMH proteins exhibits different structure compared to other 14-3-3 protein isoforms.

Yeast 14-3-3 protein isoforms BMH1 and BMH2 possess a distinctly variant C-terminal tail which differentiates them from the isoforms of higher eukaryotes. Their C-termini are longer and contain a polyglutamine stretch of unknown function. It is now well established that the C-terminal segment of 14-3-3 proteins plays an important regulatory role by functioning as an autoinhibitor which occupies the ligand binding groove and blocks the binding of inappropriate ligands. Whether the same holds true or not for the yeast isoforms is unclear. Therefore, we investigated the conformational behavior of the C-terminal segment of BMH proteins using various biophysical techniques. Dynamic light scattering, sedimentation velocity, time-resolved fluorescence anisotropy decay, and size exclusion chromatography measurements showed that the molecules of BMH proteins are significantly larger compared to the human 14-3-3zeta isoform. On the other hand, the sedimentation analysis confirmed that BMH proteins form dimers. Time-resolved tryptophan fluorescence experiments revealed no dramatic structural changes of the C-terminal segment upon the ligand binding. Taken together, the C-terminal segment of BMH proteins adopts a widely opened and extended conformation that makes difficult its folding into the ligand binding groove, thus increasing the apparent molecular size. It seems, therefore, that the C-terminal segment of BMH proteins does not function as an autoinhibitor.