Interactions and Diffusion in Fine-Stranded β-lactoglobulin Gels Determined via FRAP and Binding

The effects of electrostatic interactions and obstruction by the microstructure on probe diffusion were determined in positively charged hydrogels. Probe diffusion in fine-stranded gels and solutions of β-lactoglobulin at pH 3.5 was determined using fluorescence recovery after photobleaching (FRAP) and binding, which is widely used in biophysics. The microstructures of the β-lactoglobulin gels were characterized using transmission electron microscopy. The effects of probe size and charge (negatively charged Na₂-fluorescein (376Da) and weakly anionic 70kDa FITC-dextran), probe concentration (50 to 200 ppm), and β-lactoglobulin concentration (9% to 12% w/w) on the diffusion properties and the electrostatic interaction between the negatively charged probes and the positively charged gels or solutions were evaluated. The results show that the diffusion of negatively charged Na₂-fluorescein is strongly influenced by electrostatic interactions in the positively charged β-lactoglobulin systems. A linear relationship between the pseudo-on binding rate constant and the β-lactoglobulin concentration for three different probe concentrations was found. This validates an important assumption of existing biophysical FRAP and binding models, namely that the pseudo-on binding rate constant equals the product of the molecular binding rate constant and the concentration of the free binding sites. Indicators were established to clarify whether FRAP data should be analyzed using a binding-diffusion model or an obstruction-diffusion model.     

Electrostatics of folded and unfolded bovine β-lactoglobulin

We report on electrophoretic, spectroscopic, and computational studies aimed at clarifying, at atomic resolution, the electrostatics of folded and unfolded bovine β-lactoglobulin (BLG) with a detailed characterization of the specific aminoacids involved. The procedures we used involved denaturant gradient gel electrophoresis, isoelectric focusing, electrophoretic titration curves, circular dichroism and fluorescence spectra in the presence of increasing concentrations of urea (up to 8 M), electrostatics computations and low-mode molecular dynamics. Discrepancy between electrophoretic and spectroscopic evidence suggests that changes in mobility induced by urea are not just the result of changes in gyration radius upon unfolding. Electrophoretic titration curves run across a pH range of 3.5–9 in the presence of urea suggest that more than one aminoacid residue may have anomalous pK _a value in native BLG. Detailed computational studies indicate a shift in pKa of Glu44, Glu89, and Glu114, mainly due to changes in global and local desolvation. For His161, the formation of hydrogen bond(s) could add up to desolvation contributions. However, since His161 is at the C terminus, the end-effect associated to the solvated form strongly influences its pK _a value with extreme variation between crystal structures on one side and NMR or low-mode molecular dynamics structures on the other. The urea concentration effective in BLG unfolding depends on pH, with higher stability of the protein at lower pH.     

Quantifying Interactions of β-Synuclein and γ-Synuclein with Model Membranes

The synucleins are a family of proteins involved in numerous neurodegenerative pathologies [α-synuclein and β-synuclein (βS)], as well as in various types of cancers [γ-synuclein (γS)]. While the connection between α-synuclein and Parkinson's disease is well established, recent evidence links point mutants of βS to dementia with Lewy bodies. Overexpression of γS has been associated with enhanced metastasis and cancer drug resistance. Despite their prevalence in such a variety of diseases, the native functions of the synucleins remain unclear. They have a lipid-binding motif in their N-terminal region, which suggests interactions with biological membranes in vivo. In this study, we used fluorescence correlation spectroscopy to monitor the binding properties of βS and γS to model membranes and to determine the free energy of the interactions. Our results show that the interactions are most strongly affected by the presence of both anionic lipids and bilayer curvature, while membrane fluidity plays a very minor role. Quantifying the lipid-binding properties of βS and γS provides additional insights into the underlying factors governing the protein-membrane interactions. Such insights not only are relevant to the native functions of these proteins but also highlight their contributions to pathological conditions that are either mediated or characterized by perturbations of these interactions.     

Complexation of β-lactoglobulin fibrils and sulfated polysaccharides

Fibrils of β-lactoglobulin, formed by heating at pH 2, were titrated with a sulfated polysaccharide (κ-carrageenan) to determine the morphology and mechanism of complex formation at low pH. Structural information on the resultant complexes was gathered using transmission electron microscopy, atomic force microscopy, Doppler electrophoresis, and small-angle neutron scattering. Electrophoresis demonstrated that the carrageenan complexed with protein fibrils until reaching a maximum complexation efficiency at a protein/polysaccharide (r) weight ratio of 5:3. Neutron scattering and microscopy indicated an increasing formation of spherical aggregates attached along the protein fibrils with increases in the carrageenan concentration. These globular particles had an average diameter of 30 nm. Small-angle neutron scattering of these complexes could be accurately described by a form factor corresponding to multistranded twisted ribbons with spherical aggregates along their contour length, arranged in a necklace configuration.     

Locating the binding sites of retinol and retinoic acid with milk β-lactoglobulin

β-lactoglobulin (β-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules such as retinoids. We located the binding sites of retinol and retinoic acid on β-LG in aqueous solution at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods, and molecular modeling. The retinoid-binding sites and the binding constants as well as the effect of retinol and retinoic acid complexation on protein stability and secondary structure were determined. Structural analysis showed that retinoids bind strongly to β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K _{retinol- β -LG?}=?6.4 (±?.6)?×?10⁶?M^?1 and K _{retinoic acid- β -LG?}=?3.3 (±?.5)?×?10⁶?M^?1. The number of retinoid molecules bound per protein (n) is 1.1 (±?.2) for retinol and 1.5 (±?.3) for retinoic acid. Molecular modeling showed the participation of several amino acids in the retinoid–protein complexes with the free binding energy of ?8.11?kcal/mol for retinol and ?7.62?kcal/mol for retinoic acid. Protein conformation was altered with reduction of β-sheet from 59 (free protein) to 52–51% and a major increase in turn structure from 13 (free protein) to 24–22%, in the retinoid–β-LG complexes, indicating a partial protein destabilization.     

Locating the binding sites of retinol and retinoic acid with milk β-lactoglobulin

β-lactoglobulin (β-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules such as retinoids. We located the binding sites of retinol and retinoic acid on β-LG in aqueous solution at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods, and molecular modeling. The retinoid-binding sites and the binding constants as well as the effect of retinol and retinoic acid complexation on protein stability and secondary structure were determined. Structural analysis showed that retinoids bind strongly to β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K (retinol-) (β) (-LG?)=?6.4 (±?.6)?×?10(6)?M(-1) and K (retinoic acid-) (β) (-LG?)=?3.3 (±?.5)?×?10(6)?M(-1). The number of retinoid molecules bound per protein (n) is 1.1 (±?.2) for retinol and 1.5 (±?.3) for retinoic acid. Molecular modeling showed the participation of several amino acids in the retinoid-protein complexes with the free binding energy of -8.11?kcal/mol for retinol and -7.62?kcal/mol for retinoic acid. Protein conformation was altered with reduction of β-sheet from 59 (free protein) to 52-51% and a major increase in turn structure from 13 (free protein) to 24-22%, in the retinoid-β-LG complexes, indicating a partial protein destabilization.     

Binding of biogenic and synthetic polyamines to β-lactoglobulin

The bindings of biogenic polyamines spermine (spm), spermidine (spmd) and synthetic polyamines 3,7,11,15-tetrazaheptadecane·4HCl (BE-333) and 3,7,11,15,19-pentazahenicosane·5HCl (BE-3333) with β-lactoglobulin (β-LG) were determined in aqueous solution. FTIR, UV-vis, CD and fluorescence spectroscopic methods as well as molecular modeling were used to determine the polyamine binding sites and the effect of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind β-LG via both hydrophilic and hydrophobic contacts. Stronger polyamine-protein complexes formed with synthetic polyamines than biogenic polyamines, with overall binding constants of K_spm-β-LG = 3.2(±0.6) × 10⁴ M⁻¹, K_spmd-β-LG = 1.8(±0.5) × 10⁴ M⁻¹, K_BE-333-β-LG = 5.8(±0.3) × 10⁴ M⁻¹ and K_{BE-3333-β-LG} = 6.2(±0.05) × 10⁴ M⁻¹. Molecular modeling showed the participation of several amino acids in the polyamine complexes with the following order of polyamine-protein binding affinity: BE-3333 > BE-333 > spermine > spermidine, which correlates with their positively charged amino group content. Alteration of protein conformation was observed with a reduction of β-sheet from 57% (free protein) to 55-51%, and a major increase of turn structure from 13% (free protein) to ∼21% in the polyamine-β-LG complexes, indicating a partial protein unfolding.     

Effect of chitosan degradation on its interaction with β-lactoglobulin

Complexes between chitosan and β-lactoglobulin (β-Lg) were investigated, and their formation was found to depend on pH and ionic strength. The electrostatic attraction between the cationic polysaccharide and the negatively charged protein above its isoelectric point has been identified as the main driving force in the molecular recognition process. At low protein concentration, soluble complexes were shown to be formed, and their structural features were characterized by circular dichroism (CD) and steady-state fluorescence. Both the overall secondary structure of the protein and the local environment probed by its tryptophan residues are not affected by the presence of chitosan in the complex. Furthermore, the formation of the complex does not lead to a net stabilization of the native state of the protein over its denatured state due to formation of a similarly stable complex between the polyelectrolyte and the denatured state of the protein. The formation of coacervates between β-Lg and chitosan was also characterized as a function of average molecular weight of chitosan (subjected to ultrasonication for different periods of time: 0, 5, 15, and 30 min) by means of both turbidimetric and calorimetric techniques. The combination of turbidimetric as well as isothermal calorimetric titrations have allowed the deconvolution of two processes usually coupled in the formation of protein-polyelectrolyte coacervates: the formation of complex coacervates as the protein sites become saturated by polyelectrolyte molecules and the redissolution of the coacervates as the polyelectrolyte-to-protein ratio increases.     

Purification and characterization of β-structural domains of β-lactoglobulin liberated by limited proteolysis

Incubation of β-lactoglobulin with immobilized trypsin at 5–10°C results in a time-dependent release of several fragments of the core domain in yields approaching 15%. Digests were fractionated by ion-exchange chromatography with a Mono Q HR5/5 column and analyzed after disulfide reduction by polyacrylamide gel electrophoresis in sodium dodecylsulfate. Three fragments with approximate molecular weights of 13.8, 9.6, and 6.7 kD were identified. The fraction from ion-exchange chromatography yielding the 6.7 kD fraction after disulfide reduction was further characterized because it was most homogeneous and gave the highest yield. The C-terminal cleavage site of the 6.7 kD core fragment appeared to be Lys₁₀₀ or Lys₁₀₁ as determined by C-terminal amino acid analysis. The exact masses, after reduction with dithiothreitol, are 6195 and 6926 as determined by laser desorption mass spectrometry, corresponding to residues 48–101 and 41–100. Prior to reduction, β-lactoglobulin C-terminal residues 149–162 are connected to these core domain fragments as shown by C-terminal analysis and mass spectrometry. Structural studies indicate that these 7.9 and 8.6 kD core domain fragments released by immobilized trypsin retain much of their native structure. CD spectra indicate the presence of antiparallel β-sheet structure similar to the native protein but the α-helix is lost. Spectra in the aromatic region indicate the existence of tertiary structure. Moreover, structural transitions in urea are completely reversible as measured by CD spectra, although the extrapolated ΔG _D ^H20 and the urea concentration at the transition midpoint are lower than for the native protein. The core domain fragments also display apH-dependent binding to immobilizedtrans-retinal as does intact protein. A single endotherm is obtained for both core domain fragments and native protein upon differential scanning calorimetry, but again, the domain is less stable as indicated by a transition peak maxima of 56.9°C as compared with 81.1°C for native protein.     

Thermodynamic Study of Iodocyanopindolol β-Adrenoceptors Interactions with Rat Lung and Cerebral Cortex

Abstract

(±)¹²⁵ I-cyanopindolol (±) I CYP) was used to characterize β-adrenoceptors on rat lung and cerebral cortex membranes. The affinity of (±) ICYP was higher for lung (K^d = 64.3 pM) at 37°C. The association reaction of (±) ICYP was faster with lung (k₊₁ = 1.52 × 10⁹ M^?1.min^?1) than with cerebral cortex β-adrenoceptors (k₊₁ = 1.75 × 10⁸ M^?1.min^?1). In both tissues, the dissociation reaction followed a biphasic process with a fast (t ½ = 15.4 min and 5.6 min for lung and cerebral cortex respectively) and a slow component (t ½ = 474 min and 255 min for lung and cerebral cortex respectively). The thermodynamic parameters for (±) ICYP - β-adrenoceptors binding have been determined from kinetics and equilibrium studies, for the two tissues, at several temperatures between 0° and 44° C. For lung and cerebral cortex, Arrhenius plots were linear with different energies of activation. Van't Hoff plot was not linear for lung and the standard enthalpy and entropy changes of (±) ICYP - β-adrenoceptors interaction decreased linearly with temperature : the binding occured with a negative heat capacity change (ΔCp° = -368.9 cal. moles^?1. K^?1) at 25° C. Thermodynamic and kinetic results show that binding of (±) ICYP to lung β-adrenoceptors could involve two successive equilibria with a conformational change of the β-adrenergic receptor.     

Ectopic expression of β-lactoglobulin transgenes

Genomic constructs comprising the ovine β-lactoglobulin gene are expressed in a position-independent manner in the mammary gland of transgenic mice. In some lines however, constitutive low-level transgene expression was detected in all other tissues. This ectopic expression presumably represents a position-dependent phenomenon since it was observed in only a proportion (40%) of the lines generated. Different lines of BLG transgenic mice displayed similar temporal patterns of ectopic expression. This pattern differed from that of BLG in the mammary gland. These data imply that the DNA elements that direct position-independent expression of β-lactoglobulin transgenes in the mammary gland do not have the ability to insulate them from position effects in other tissues. Furthermore, the relatively high frequency and constitutive nature of ectopic expression suggests that transgene integration may not be totally random.     

Solubility and reactivity of caseins and β-lactoglobulin in protic solvents

The study of the solubility of unstructured proteins (α_S1-, β-, and κ-casein) and well-structured globulin (β-lactoglobulin) in low water binary solvent systems demonstrated the crucial importance of solvent polarity and neutralization of protein polar functions on the final outcome of solubility experiments. The solubilities up to 38, 56, and 96% in CHCl₃/CH₃OH (1/1, v/v) acidified with HCl and up to 5, 10, and 25% in CHCl₃/CH₃OH (1/1, v/v) in the presence of triethylamine (TEA) were obtained for κ-, α_S1-, and β-casein, respectively. The importance of protein charge neutralization was apparent when the solubilization was performed in basified CHCl₃/CH₃OH media, giving the optimal results when the studied proteins were brought before to their isoionic point. The maximum solubility of β-casein at its pI in 30–70% methanol in CHCl₃ was reaching 50–60% with triethylamine (TEA) added. β-lactoglobulin could be solubilized up to 70% in CHCl₃/CH₃OH (7/3, v/v) acidified with HCl and up to 40% in CHCl₃/CH₃OH (3/7, v/v) in the presence of TEA. The observed yield of reductive alkylation of β-lactoglobulin was much higher (98%) when performed in studied solvent system than in aqueous conditions (75%). Apparently, steric hindrance of the well-folded β-barrel (in aqueous conditions) structure masks the portion of ε-NH₂ groups. In the case of unstructured aqueous media β-casein, 90% alkylation yields were obained in organic and aqueous conditions.     

Probing the binding sites of resveratrol,genistein, and curcumin with milk β-lactoglobulin

We determined the binding sites of curcumin (cur), resveratrol (res), and genistein (gen) with milk β-lactoglobulin (β-LG) at physiological conditions. Fourier transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of polyphenol–protein complexes. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K_{curcumin–β-LG}?=?4.4 (±?.4)?×?10⁴ M^?1, K_{resveratrol–β-LG}?=?4.2 (±?.2)?×?10⁴ M^?1, and K_{genistein–β-LG}?=?1.2 (±?.2)?×?10⁴?M^?1. The number of polyphenol molecules bound per protein (n) was 1 (cur), 1.1 (res), and 1 (gen). Molecular modeling showed the participation of several amino acid residues in polyphenol–protein complexation with the free binding energy of ?12.67 (curcumin–β-LG), ?12.60 (resveratrol–β-LG), and ?10.68?kcal/mol (genistein–β-LG). The order of binding was cur?>?res?>?gen. Alteration of the protein conformation was observed in the presence of polyphenol with a major reduction of β-sheet and an increase in turn structure, causing a partial protein structural destabilization. β-LG might act as a carrier to transport polyphenol in vitro.     

Structure and stability of Gyuba, a β-lactoglobulin chimera

β-lactoglobulin (LG) contains nine β-strands (strands A-I) and one α-helix. Strands A-H form a β-barrel. At neutral pH, equine LG (ELG) is monomeric, whereas bovine LG (BLG) is dimeric, and the I-strands of its two subunits form an intermolecular β-sheet. We previously constructed a chimeric ELG in which the sequence of the I-strand was replaced with that of BLG. This chimera did not dimerize. For this study, we constructed the new chimera we call Gyuba (which means cow and horse in Japanese). The amino acid sequence of Gyuba includes the sequences of the BLG secondary structures and those of the ELG loops. The crystal structure of Gyuba is very similar to that of BLG and indicates that Gyuba dimerizes via the intermolecular β-sheet formed by the two I-strands. Thus, the entire arrangement of the secondary structural elements is important for LG dimer formation.     

Characteristics and effects of specific peptides on heat-induced aggregation of β-lactoglobulin

A bovine β-lactoglobulin hydrolysate, obtained by the hydrolysis by the Glu specific enzyme Bacillus licheniformis protease (BLP), was fractionated at pH 7.0 into a soluble and an insoluble fraction and characterized by LC-MS. From the 26 peptides identified in the soluble fraction, five peptides (A[f97-112] = [f115-128], AB[f1-45], AB[f135-157], AB[f135-158], and AB[f138-162]) bound to β-lactoglobulin at room temperature. After heating of β-lactoglobulin in the presence of peptides, eight peptides were identified in the pellet formed, three of them belonging to the previously mentioned peptides. Principle component analysis revealed that the binding at room temperature (to β-lactoglobulin) was related to the total hydrophobicity and the total charge of the peptides. The binding to the unfolded protein could not be attributed to distinct properties of the peptides. The presence of the peptides caused a 50% decrease in denaturation enthalpy (from 148 ± 3 kJ/mol for the protein alone to 74 ± 2 kJ/mol in the presence of peptides), while no change in secondary structure or denaturation temperature was observed. At temperatures <85 °C, the addition of peptides resulted in a 30-40% increase of precipitated β-lactoglobulin. At pH < 6, no differences in the amount of aggregated β-lactoglobulin were observed, which indicates the lack of binding of peptides to β-lactoglobulin at those pH values as was also observed by SELDI-TOF-MS. Although only a few peptides were found to participate in aggregation, suggesting specificity, principal component analysis was unable to identify specific properties responsible for this.     

Thermodynamic and structural analysis of homodimeric proteins: Model of β-lactoglobulin

The energetics of protein homo-oligomerization was analyzed in detail with the application of a general thermodynamic model. We have studied the thermodynamic aspects of protein-protein interaction employing β-lactoglobulin A from bovine milk at pH = 6.7 where the protein is mainly in its dimeric form. We performed differential calorimetric scans at different total protein concentration and the resulting thermograms were analyzed with the thermodynamic model for oligomeric proteins previously developed. The thermodynamic model employed, allowed the prediction of the sign of the enthalpy of dimerization, the analysis of complex calorimetric profiles without transitions baselines subtraction and the obtainment of the thermodynamic parameters from the unfolding and the association processes and the compared with association parameters obtained with Isothermal Titration Calorimetry performed at different temperatures. The dissociation and unfolding reactions were also monitored by Fourier-transform infrared spectroscopy and the results indicated that the dimer of β-lactoglobulin (N₂) reversibly dissociates into monomeric units (N) which are structurally distinguishable by changes in their infrared absorbance spectra upon heating. Hence, it is proposed that β-lactoglobulin follows the conformational path induced by temperature:N₂ ? 2N ? 2D. The general model was validated with these results indicating that it can be employed in the study of the thermodynamics of other homo-oligomeric protein systems.     

Thermodynamic and structural analysis of homodimeric proteins: model of β-lactoglobulin

The energetics of protein homo-oligomerization was analyzed in detail with the application of a general thermodynamic model. We have studied the thermodynamic aspects of protein-protein interaction employing β-lactoglobulin A from bovine milk at pH=6.7 where the protein is mainly in its dimeric form. We performed differential calorimetric scans at different total protein concentration and the resulting thermograms were analyzed with the thermodynamic model for oligomeric proteins previously developed. The thermodynamic model employed, allowed the prediction of the sign of the enthalpy of dimerization, the analysis of complex calorimetric profiles without transitions baselines subtraction and the obtainment of the thermodynamic parameters from the unfolding and the association processes and the compared with association parameters obtained with Isothermal Titration Calorimetry performed at different temperatures. The dissociation and unfolding reactions were also monitored by Fourier-transform infrared spectroscopy and the results indicated that the dimer of β-lactoglobulin (N(2)) reversibly dissociates into monomeric units (N) which are structurally distinguishable by changes in their infrared absorbance spectra upon heating. Hence, it is proposed that β-lactoglobulin follows the conformational path induced by temperature:N(2)?2N?2D. The general model was validated with these results indicating that it can be employed in the study of the thermodynamics of other homo-oligomeric protein systems.     

Connexin-43 Interactions with ZO-1 and α- and β-tubulin

Gap junctions are composed of connexins that form transmembrane channels between adjacent cells. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating. Interestingly, channel-independent processes regulated by Cx43 have also been postulated. In our studies to elucidate the mechanism of Cx43 channel gating by growth factors and to explore additional functions of gap junctions, we have identified three interacting partners of the C-terminal tail of Cx43 (Cx43CT). (i) the c-Src tyrosine kinase, which phosphorylates Cx43CT and is involved in G protein-mediated inhibition of Cx43 gap junctional communication, (ii) the ZO-1 ‘scaffold’ protein, which might recruit signaling proteins into Cx43-based gap junctions. (iii) microtubules (consisting of α/β-tubulin dimers), which extend with their distal ends to Cx43-based gap junctions, suggesting that Cx43 gap junctions may play a novel role in regulating microtubule stability in contacted cells. Here we show that Cx43 binds α-tubulin equally well as β-tubulin. In addition, we show that the second, but not the first, PDZ domain of ZO-1 binds directly to Cx43, and we confirm that the very C-terminal isoleucine residue of Cx43 is critical for ZO-1 binding.     

Enzymatic modification of β-lactoglobulin with N-fatty-acyl-dipeptide by transglutaminase from Streptomyces mobaraense

-Lactoglobulin was enzymatically acylated with N-lauroyl-l-glutaminyl-glycine and N-lauroyl-l-glutamyl-l-lysine using transglutaminase from Streptomyces mobaraense. The modification of the protein with N-fatty-acyl-dipeptide was confirmed by SDS-PAGE, gel chromatography, HPLC, amino acid analysis, and TOF-MS. The degrees of the protein modification with N-lauroyl-l-glutaminyl-glycine and N-lauroyl-l-glutamyl-l-lysine were estimated to be 2–4 and 1.5 residues per molecule, respectively.