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.     

Differences in heat stability and ligand binding among β-lactoglobulin genetic variants A,B and C using H NMR and fluorescence quenching

The structure of β-lactoglobulin (β-LG) is well characterized, but the exact location of binding sites for retinol and (−)-epigallocatechingallate (EGCG) is still a subject of controversy. Here we report that the genetic β-LG variants A, B and C have different numbers of binding sites for retinol (almost completely incorporated into the calyx), as well as for EGCG (exclusively bound on the surface), and β-LG A with the most binding sites for EGCG, which include Tyr²⁰, Phe¹⁵¹ and His⁵⁹. Upon heat related unfolding, new unspecific binding sites emerge, which are comparable in number and affinity for retinol and for EGCG, and in the three genetic variants A, B and C. The findings of our study provide new insights into the use of β-LG as nanotransporter.     

A study of the effect of sodium dodecyl sulfate on bovine β-casein self-association

The effect of low concentrations of sodium dodecyl sulfate on the self-association of β-casein in solution has been reinvestigated at neutral pH by using instrinsic fluorescence measurements, analytical ultracentrifugation, gel filtration chromatography, and the fluorescent properties of the probe, anilinonaphthalene sulfonate. Sodium dodecyl sulfate was found to interact with the protein so that the normal equilibrium between monomers and micellelike polymers was displaced toward polymer formation. At higher concentrations of sodium dodecyl sulfate, the β-casein polymers became smaller while the monomer-polymer equilibrium remained displaced toward polymer formation. It seems likely that there is a limited number of sites on the β-casein molecule that bind sodium dodecyl sulfate strongly. As a consequence of this binding, the balance of electrostatic and hydrophobic forces is altered to increase the degree of self-association at low concentrations of sodium dodecyl sulfate, despite the increase in net negative charge per protein monomer.     

Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin

Proteolytic digestion of bovine β-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15–20), AASDISLLDAQSAPLR (residues 25–40), IPAVFK (residues 78–83) and VLVLDTDYK (residues 92–100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55–64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of β-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of β-lactoglobulin could be useful to increase its antimicrobial function.     

Important role of methionine 145 in dimerization of bovine β-lactoglobulin

β-Lactoglobulin (LG) contains nine β-strands (strands A-I) and one α-helix. Strands A-H form a β-barrel. At neutral pH, bovine LG (BLG) forms a dimer and the dimer interface consists of AB-loops and the I-strands of two subunits. On the other hand, equine LG (ELG) is monomeric. The residues 145-153 of BLG, which compose a dimer interface, are entirely different from those of ELG. The difference in the association states between BLG and ELG can be attributed to the residues 145-153. To confirm this, we constructed a chimeric LG, ImBLG (I-strand mutated BLG), in which the residues 145-153 were replaced with those of ELG. Gel-filtration chromatography and analytical ultracentrifugation revealed that ImBLG existed as a monomer. To identify the residues important for dimerization, we constructed several revertants and investigated their association. This experiment revealed that, in addition to the interface residues (Ile147, Leu149 and Phe151), Met145 is critical for dimerization. Although Met145 does not contact with the other protomer, it seems to be important in determining the backbone conformation of the I-strand. This was supported by the fact that all Met145-containing mutants showed circular dichroism spectra similar to BLG but different from ImBLG.     

Structural and thermodynamic studies of binding saturated fatty acids to bovine β-lactoglobulin

Lactoglobulin is a globular milk protein for which physiological function has not been clarified. Due to its binding properties lactoglobulin might serve as a carrier for bioactive molecules. Binding of 12-, 14-, 16- and 18-carbon saturated fatty acids to bovine β-lactoglobulin has been characterised by isothermal titration calorimetry and X-ray crystallography as a part of systematic studies of lactoglobulin complexes with ligands of biological importance. The thermodynamic parameters have been determined for lauric, myristic and palmitic acid complexes revealing systematic decrease of enthalpic and increase of entropic component of ΔG with elongation of aliphatic chain. In all crystal structures determined with resolution 1.9-2.1?, single fatty acid molecule was found in the β-barrel in extended conformation with individual pattern of interactions. Location of a fatty acid in the binding site depends on the length of aliphatic chain and influences polar interactions between protein and ligand. Systematic changes of entropic component indicate important role of water in binding process.     

A comparison study of the interaction between β-lactoglobulin and retinol at two different conditions: spectroscopic and molecular modeling approaches

The interaction between bovin β-Lactoglobulin (β-LG) and retinol at two different pH values was investigated by multispectroscopic, zeta potential, molecular modeling, and conductometry measurements. The steady state and polarization fluorescence spectroscopy revealed that complex formation at two different pH values could occur through a remarkable static quenching. According to fluorescence quenching, one set of binding site at pH 2 and two sets of binding sites at pH 7 were introduced for binding of retinol to β-LG that show the enhancement of saturation score of β-LG to retinol in dimmer condition. The polarization fluorescence analysis represented that there is more affinity between β-LG and retinol at pH 7 rather than at pH 2. The effect of retinol on β-LG was studied by UV-visible, circular dichroism (CD), and synchronous fluorescence, which indicated that retinol induced more structural changes on β-LG at pH 7. β-LG–retinol complex formation at two different pH values was recorded via applying resonance light scattering (RLS) and zeta potential. Conductometry and RLS showed two different behaviors of interaction between β-LG and retinol at two different pH values; therefore, dimmer formation played important roles in different behaviors of interaction between β-LG and retinol. The zeta potential was the implied combination of electrostatic and hydrophobic forces which are involved in β-LG–retinol complex at two different pH values, and the hydrophobic interactions play a dominant role in complex formation. Molecular modeling was approved by all experimental results. The acquired results suggested that monomer and dimmer states of β-LG can be induced by retinol with different behaviors.     

Affinity chromatography of bovine trypsin. A rapid separation of bovine α- and β-trypsin

Affinity adsorbents for bovine trypsin were prepared by covalently coupling p-(p′-amino-phenoxypropoxy)benzamidine to cellulose and to agarose. Trypsin binds to both adsorbents at pH6–8 and is released at low pH values or in the presence of n-butylamine hydrochloride. Pure β-trypsin may be eluted from crude trypsin bound at pH8.0 to the cellulose adsorbent by stepwise elution with an acetate buffer, pH5.0. Both α- and β-trypsin may be isolated by chromatography of crude trypsin on the agarose derivative in an acetate buffer, pH4.0. These two methods for purifying the trypsin are specific to the particular adsorbents. They are rapid and convenient in use. Both methods leave a mixture of the two enzymes bound to the adsorbent and release occurs only at low pH values. The effects of pH, composition and ionic strength of buffer and other variables on both purification methods are described. Affinity adsorbents of soya-bean trypsin inhibitor and of N-α-(N′-methyl-N′-sulphanilyl) sulphanilylagmatine bound to agarose were prepared, but were found to be of limited usefulness in the purification of trypsin.     

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.     

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.     

Structural and conformational changes of β-lactoglobulin B: an infrared spectroscopic study of the effect of pH and temperature

Infrared spectra of 2.5 mM solutions of β-lactoglobulin B were recorded as a function of pH (from pH 2 to pH 13) and as a function of temperature (from −100°C to +90°C). An analysis of the pH- and temperature-induced changes in the secondary structures was performed based on changes in the conformation-sensitive amide I bands of β-lactoglobulin. Whereas the total of β-structure remains constant (56–59%) between pH22 and pH 10, the proportions of the various β-components do change. In particular, the dimerization of the monomeric protein, induced by raising the pH from 20 to 3, leads to an increase in the intensity of the 1636 cm⁻¹ band (associated with antiparallel β-sheet), at the expense of the 1626 cm⁻¹ band (associated with exposed β-strands). Both the thermal and alkaline denaturation of β-lactoglobulin occur in two distinct stages. Although the spectra (i.e., the structures) after complete thermal or alkaline denaturation are clearly different, the spectrum of the protein after the first stage of thermal denaturation (at about 60°C) is the same as that after the first stage of alkaline denaturation (at pH 11), suggesting a common denaturation intermediate, which probably represents a crossover point in a complex potential hypersurface.     

Evaluation of temperature effect on the interaction between β-lactoglobulin and anti-β-lactoglobulin antibody by atomic force microscopy

Molecular recognition such as antigen-antibody interaction is characterized by the parameters of kinetics and the energy landscape. Examinations of molecules involved in the interaction at different temperatures using atomic force microscopy (AFM) can provide information on not only the effects of temperature on the unbinding force between a molecule of interest and a complementary molecule but also the parameters of kinetics and the energy landscape for dissociation of the molecular complex. We investigated the effect of temperature on the dissociation process of the complex of β-lactoglobulin and anti-bovine β-lactoglobulin IgG polyclonal antibody using AFM. Measurements of the unbinding forces between β-lactoglobulin and the antibody were performed at 25, 35, and 45 °C. The following results were obtained in our present study: (i) The unbinding forces decreased as temperature increased, suggesting that the binding force between β-lactoglobulin and the antibody includes the force originating from temperature-dependent interactions (e.g., hydrogen bonding). (ii) At each temperature, the unbinding force exhibited two linear regimes in the force spectra, indicating that the dissociation process of the β-lactoglobulin-antibody complex passes at least two energy barriers from the bound state to the dissociated state. (iii) The dissociation rates at zero force and the position of energy barriers increased as temperature increased. (iv) The heights of the two energy barriers in the reaction coordinates were 49.7 k(B)T and 14.5 k(B)T. (v) The values of roughness of the barriers were ca. 6.1 k(B)T and 3.2 k(B)T. Overall, the present study using AFM revealed more information about the β-lactoglobulin-antibody interaction than studies using conventional bulk measurement such as surface plasmon resonance.     

The interrelationship between ligand binding and self-association of the folate binding protein. The role of detergent–tryptophan interaction

Background

The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of folate binding.

Methods

Self-association behavior of apo- and holo-FBP was addressed through size exclusion chromatography, SDS-PAGE, mass spectrometry, surface plasmon resonance and fluorescence spectroscopy.

Results

Especially holo-FBP exhibits concentration-dependent self-association at pH 7.4 (pI), and is more prone to associate into stable complexes than apo-FBP. Even more pronounced was the tendency to complexation between apo-FBP and holo-FBP in accord with a model predicting association between apo and holo monomers [19]. This will lead to removal of apo monomers from the reaction scheme resulting in a weak incomplete ligand binding similar to that observed at FBP concentrations < 10 nM. The presence of synthetic and natural detergents normalized folate binding kinetics and resulted in appearance of monomeric holo-FBP. Fluorescence spectroscopy indicated molecular interactions between detergent and tryptophan residues located in hydrophobic structures of apo-FBP which may participate in protein associations.

General significance

Self-association into multimers may protect binding sites, and in case of holo-FBP even folate from biological degradation. High-affinity folate binding in body secretions, typically containing 1–10 nM FBP, requires the presence of natural detergents, i.e. cholesterol and phospholipids, to avoid complexation between apo- and holo-FBP.     

Interactions of β-lactoglobulin with serotonin and arachidonyl serotonin

β‐Lactoglobulin (β‐LG) is a lipocalin, which is the major whey protein of cow's milk and the milk of other mammals. However, it is absent from human milk. The biological function of β‐LG is not clear, but its potential role in carrying fatty acids through the digestive tract has been suggested. β‐LG has been found in complexes with lipids such as butyric and oleic acids and has a high affinity for a wide variety of compounds. Serotonin (5‐hydroxytryptamine, 5‐HT), an important compound found in animals and plants, has various functions, including the regulation of mood, appetite, sleep, muscle contraction, and some cognitive functions such as memory and learning. In this study, the interaction of serotonin and one of its derivatives, arachidonyl serotonin (AA‐5HT), with β‐LG was investigated using circular dichroism (CD) and fluorescence intensity measurements. These two ligands interact with β‐LG forming equimolar complexes. The binding constant for the serotonin/β‐LG interaction is between 10⁵ and 10⁶ M⁻¹, whereas for the AA‐5HT/β‐LG complex it is between 10⁴ and 10⁵ M⁻¹ as determined by measurements of either protein or ligand fluorescence. The observed binding affinities were higher in hydroethanolic media (25% EtOH). The interactions between serotonin/β‐LG and AA‐5HT/β‐LG may compete with self‐association (micellization) of both the ligand and the protein. According to far‐ and near‐UV CD results, these ligands have no apparent influence on β‐LG secondary structure, however they partially destabilize its tertiary structure. Their binding by β‐LG may be one of the peripheral mechanisms of the regulation of the content of serotonin and its derivatives in the bowel of milk‐fed animals. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 871–880, 2011.     

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.     

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.     

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.