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.     

Analysis of various indefinite self-associations

Two methods have been developed for the analysis of four types of indefinite self-associations. Unlike previous treatments by others, the procedures can be applied to nonideal cases. The two methods were first tested with simulated data. and it was found that one could indeed distinguish between the four types of indefinite self-associations. For a more realistic test, sedimentation equilibrium experiments were performed on solutions of β-lactoglobulin A at 16°C in 0.15 ionic strength acetate buffer, pH 4.65. The self-association of the β-lactoglobulin A was best described by either method as a sequential indefinite self-association having two equilibrium constants and one second virial coefficient.     

The temperature-dependent self-association of beta-lactoglobulin C in glycine buffers

The self-association of β-lactoglobulin C at low pH (ca. 2.5) in glycine buffers has been studied at four temperatures, 10, 16, 20, and 25 °C, by low- and high-speed sedimentation equilibrium experiments. One buffer had an ionic strength of 0.1 and the other an ionic strength of 0.2. With either buffer the concentration dependence of the apparent weight average molecular weight, M_wa, was characteristic of a nonideal self-association. Like its genetic variants, β-lactoglobulin A and B, the self-association of β-lactoglobulin C increased with decreasing temperature; however, at the same temperature the association was always stronger in the buffer having the higher ionic strength. Several models were used to test the self-association, and a monomer-dimer self-association seemed to describe the self-association best with either buffer. Values of the association equilibrium constant, K₂, and the second virial coefficient, BM₁, are reported at each temperature for both series of experiments. Values of the thermodynamic functions, ΔG °, ΔH °, and ΔS °, are also reported for these experiments.     

The effect of SDS on protein zone dispersion in polyacrylamide gel electrophoresis

The zone dispersions of the reduced subunit of β-lactoglobulin B and its derivative with sodium dodecyl sulfate (SDS) were measured during polyacrylamide gel electrophoresis (PAGE) using the apparatus for continuous optical scanning at 280 nm. The ratio of apparent diffusion coefficients (D′) of the reduced subunit of β-lactoglobulin B (1.71 × 10^?6 cm²/s) and of its SDS-derivative (7.1 × 10^?7 cm²/s) was found to be 2.4 under the conditions of PAGE (pH 10.4, 0.015 ionic strength, 1°C, 4 mA/cm² current density, 50 μg protein load, 10% T gel) used. This is nearly twice the value of 1.3 predicted, under the assumption of sphericity for these protein molecules, on the basis of the binding of 1.4 g of SDS per gram of protein. It is postulated that the increment in zone sharpness (decrease in apparent diffusion coefficient) over that predicted by SDS binding alone is a general property of SDS-proteins providing gel electrophoresis in SDS-containing buffers with a resolving power larger than that obtained in the absence of the detergent.     

The Effect of pH and Heat Pre-Treatments on the Physicochemical and Emulsifying Properties of β-lactoglobulin

The overall goal of this research was to investigate structure-function mechanisms associated the emulsifying properties β-lactoglobulin (β-LG). Specifically the physicochemical (i.e., surface charge and hydrophobicity, size and interfacial tension) and emulsifying (i.e., emulsification activity (EAI) and stability indices (ESI)) properties of β-LG were investigated in response to changes in pH (3.0, 5.0 and 7.0) and heat pre-treatment conditions (25, 55 and 85 °C). Hydrophobicity was found to be greatest at pH 5.0/85 °C, whereas at all conditions it was significantly lower. Surface charge on β-LG was found to be neutral at?~?pH 3.9, regardless of conditions. Aggregate size was also found to be highest at pH 5.0/85 °C (avg. hydrodynamic radii of ~714 nm), corresponding to a reduced net surface charge and high hydrophobicity. Little size dependence of aggregates was observed at pH 3.0 regardless to the temperature pre-treatments (radii ~120 nm). In contrast, at pH 7.0 slight temperature dependence was apparent, where treatments at 25, 55 and 85 °C led to radii of 412.8, 307.2 and 232.3 nm, respectively. Overall, the addition of β-LG to a canola oil–water system resulted in a decline in interfacial tension from ~28 mN/m to ~18 mN/m, however the effect of pH/temperature conditions was minimal. EAI was found to be highest when β-LG solutions displayed high surface charge combined with moderate hydrophobicity. In contrast, ESI was higher under conditions where β-LG solutions remained in a native (25 °C) or fully denatured state (85 °C) versus one in where partially unravelling may be occurring (55 °C).     

Characterisation of a novel Bacillus sp. SJ-10 β-1,3–1,4-glucanase isolated from jeotgal, a traditional Korean fermented fish

A novel β-1,3–1,4-glucanase gene was identified in Bacillus sp. SJ-10 (KCCM 90078) isolated from jeotgal, a traditional Korean fermented fish. We analysed the β-1,3–1,4-glucanase gene sequence and examined the recombinant enzyme. The open reading frame of the gene encoded 244 amino acids. The sequence was not identical to any β-glucanases deposited in GenBank. The gene was cloned into pET22b(+) and expressed in Escherichia coli BL21. Purification of recombinant β-1,3–1,4-glucanase was conducted by affinity chromatography using a Ni-NTA column. Enzyme specificity of β-1,3–1,4-glucanase was confirmed based on substrate specificity. The optimal temperature and pH of the purified enzyme towards barley β-glucan were 50 °C and pH 6, respectively. More than 80 % of activity was retained at temperatures of 30–70 °C and pH values of 4–9, which differed from all other bacterial β-1,3–1,4-glucanases. The degradation products of barley β-glucan by β-1,3–1,4-glucanase were analysed using thin-layer chromatography, and ultimately glucose was produced by treatment with cellobiase.     

Surface-directed structure formation of β-lactoglobulin inside droplets

The morphology of β-lactoglobulin structures inside droplets was studied during aggregation and gelation using confocal laser scanning microscopy (CLSM) equipped with a temperature stage and transmission electron microscopy (TEM). The results showed that there is a strong driving force for the protein to move to the interface between oil and water in the droplet, and the β-lactoglobulin formed a dense shell around the droplet built up from the inside of the droplets. Less protein was found inside the droplets. The longer the β-lactoglobulin was allowed to aggregate prior to gel formation, the larger the part of the protein went to the interface, resulting in a thicker shell and very little material being left inside the droplets. The droplets were easily deformed because no network stabilizes them. When 0.5% emulsifier, polyglycerol polyresinoleat (PGPR), was added to the oil phase, the β-lactoglobulin was situated both inside the droplets and at the interface between the droplets and the oil phase; when 2% PGPR was added, the β-lactoglobulin structure was concentrated to the inside of the droplets. The possibility to use the different morphological structures of β-lactoglobulin in droplets to control the diffusion rate through a β-lactoglobulin network was evaluated by fluorescence recovery after photobleaching (FRAP). The results show differences in the diffusion rate due to heterogeneities in the structure: the diffusion of a large water-soluble molecule, FITC-dextran, in a dense particulate gel was 1/4 of the diffusion rate in a more open particulate β-lactoglobulin gel in which the diffusion rate was similar to that in pure water.     

Purification and characterization of an extracellular chitosanase produced by Amycolatopsis sp. CsO-2

Extracellular chitosanase produced by Amycolatopsis sp. CsO-2 was purified to homogeneity by precipitation with ammonium sulfate followed by cation exchange chromatography. The molecular weight of the chitosanase was estimated to be about 27,000 using SDS-polyacrylamide gel electrophoresis and gel filtration. The maximum velocity of chitosan degradation by the enzyme was attained at 55°C when the pH was maintained at 5.3. The enzyme was stable over a temperature range of 0–50°C and a pH range of 4.5–6.0. About 50% of the initial activity remained after heating at 100°C for 10 min, indicating a thermostable nature of the enzyme. The isoelectric point of the enzyme was about 8.8. The enzyme degraded chitosan with a range of deacetylation degree from 70% to 100%, but not chitin or CM-cellulose. The most susceptible substrate was 100% deacetylated chitosan. The enzyme degraded glucosamine tetramer to dimer, and pentamer to dimer and trimer, but did not hydrolyze glucosamine dimer and trimer.     

Physical properties of type i collagen in solution: Structure of α-Chains and β- and γ-components and two-component mixtures

The structure of thermally denatured Type I collagen has been studied using laser light scattering. The results indicate that the diffusion coefficients of α-chains and β- and γ-components are 1.550 ± 0.08 × 10^?7, 1.000 ± 0.05 × 10^?7, and 0.835 ± 0.04 × 10^?7 cm²/sec, respectively, at temperatures between 20 and 40°C. It is concluded from diffusion data that these species have hydrodynamic radii of about 13.8 nm (α-chain), 21.5 nm (β-component), and 25.7 nm (γ-component), consistent with previous studies of thermal denaturation by light scattering. It is also concluded, based on volume calculations, that a large volume increase occurs when the triple helix unfolds. Homodyne correlation functions for two component mixtures of α-chains and β-and γ-components appeared to decay exponentially. In all but one case discussed the correlation function could be fitted with a single component having a translational diffusion coefficient which was an intensity weighted average of the diffusion coefficient of each component present.     

Temperature dependence of partial volumes of proteins

The change of the apparent partial specific volumes of egg albumin, bovine serum albumin, bovine methemoglobin, β-lactoglobulin, and lysozyme with temperature through the thermal transitions of the proteins have been measured with dilatometers. Four regions in the plot of the apparent partial specific volumes against temperature can be recognized: (1) linear sections extending from 25°C up to 45–50°C: (2) a decrease in slope between 50°C and 60°C; (3) a sharp increase in slope with increasing temperature coinciding with the appearance of heat coagulation of the protein and followed by (4) a decrease in the slope. The return of the protein samples to 25°C yields linear relations between the apparent partial specific volumes of the heat-denatured proteins and the decreasing temperature.     

Conformation similarities of the globular and tailed forms of acetylcholinesterase from Torpedo california

The conformation of the globular dimer (G₂), the tailed asymmetric dodecamer (A₁₂, also containing some tailed octamer A₈) and the globular tetramer (G₄, prepared by removing the collagen-like tail from A₁₂) of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) was studied by circular dichroism (CD) in the ultraviolet region. The G₂ and G₄ forms had similar conformation with about 40% α-helix, 35% β-sheets and 4% β-turns; the tailed form had a lower helicity (about 34%) and β-form (about 25%) content probably because of the presence of the tail whose CD spectrum resembles that of an unordered form, but it had about the same amount of β-turns as the other two forms. All three forms also had similar CD spectra in the near-ultraviolet region due to their non-peptide chromophores. The pH, thermal and urea denaturation of the three acetylcholinesterase forms was also similar to each other. The pH-dependency of both the enzymatic activity and CD intensity of the three forms showed bell-shaped curves with a plateau at pH 7–8. The activity was completely lost at pH below 5 or above 10, but the corresponding CD spectra retained 70–80% of the original magnitudes. Thermal denaturation of the three forms at pH 7.5 showed a conformational transition and loss of activity between 30 and 40°C, but the CD intensity of the helical band at 222 nm was reduced by only 20–30%. Urea denaturation of the three form began at 1 M urea; it was protein concentration- and time-dependent. Again, the activity disappeared faster than the decreasing CD intensity. Thus, the overall conformation of the three acetylcholinesterase forms appears to be relatively stable, but their active site is easily perturbed by changing the environment. The loss of activity correlated well with the disapperance of the CD band of tryptophan(s) in the near-ultraviolet region, suggesting that the Trp residue(s) might be at or near the active center of the enzyme.     

Interaction of beta-lactoglobulin and cytochrome c: complex formation and iron reduction.

β-Lactoglobulin forms a soluble complex with cytochrome c in mildly alkaline solutions of low ionic strength. Sedimentation velocity experiments suggest that the complex (maximum s₂₀ = 3.7) consists of one cytochrome c molecule per β-lactoglobulin monomer unit. At pH 8 or higher, the presence of β-lactoglobulin causes reduction of ferri- to ferrocytochrome c. The initial rate of reduction at a single temperature depends primarily on the concentration of β-lactoglobulin, although the final percentage ferrocytochrome c obtained is constant at molar ratios of three or more β-lactoglobulin monomers to one cytochrome c molecule. The temperature dependence of the initial rate of iron reduction resembles that for alkaline denaturation of β-lactoglobulin. The displacement of N-dansylaziridine, a sulfhydryl specific dye, from bovine β-lactoglobulin during iron reduction, and the formation of nonreducing complexes between the analogous swine protein (no sulfhydryls) and cytochrome c suggest that the sulfhydryl group of β-lactoglobulin is the electron donor.     

ESR study of aqueous dispersions of β-lactoglobulin and spin-labelled glyceryl monostearate

From the ESR spectra of aqueous dispersions of synthetic glyceryl monostearate (spin labelled at C-12) a critical micelle concentration of 30 μmol/l at room temperature was obtained, which agrees with that deduced from surface tension measurements.At monoglyceride concentrations smaller or larger than the critical micelle concentration, the monomers show increased motional restriction with increasing molar ratio of β-latoglobulin to monoglyceride up to a value of 10, as determined from calculated rotational correlation times.A similar progressive interaction was deduced from spectral changes observed on equimolar dispersions of β-lactoglobulin and monoglyceride on raising the temperature to 55°C at which the protein and monoglyceride coprecipitate.The relevance of these findings for non-labelled monoglyceride dispersions is indicated by the similarity of the pH-dependent flocculation behaviour of labelled and non-labelled monoglycerides, both in the absence and presence of β-lactoglobulin. In addition, proton magnetic resonance and mechanical stability measurements suggest that spin-labelled glyceryl monostearate behaves analogously to non-labelled glyceryl monooleate.     

Structure of bovine beta-lactoglobulin at 6A resolution.

Bovine β-lactoglobulin is a dimer with a molecular weight of 2 × 18,400. In solution it undergoes a pH-dependent transition at pH 7.0 between two alternative structures, named N and R. The structures of four different crystal forms have been determined by multiple isomorphous replacement with heavy-atoms. Two of them, lattices K and X, were crystallised at pH 6.5, corresponding to the N state in solution; and the other two, lattices Y and Z, were crystallised at pH 7.5, corresponding to the R state in solution. The figures of merit of the phase angles determined for these lattices were 0.76, 0.77, 0.80 and 0.80, respectively. The four structures that emerged are similar and show certain features suggestive of α-helices and pleated sheets, but the resolution is insufficient to trace the entire course of the polypeptide chain. No clear distinction can yet be made between the structures above or below pH 7.0, nor between the native molecule and the molecule from which the C-terminal leucine and histidine residues have been cleaved. Analyses at higher resolution are in progress.     

The effect of high pressure on the rates of proteolytic hydrolysis,II. Trypsin

The interpretation of the volume change of activation for varying conditions of proteolytic hydrolysis is discussed.A volume change of activation of ?36 ml. was calculated from the acceleration of the rate of hydrolysis of β-lactoglobulin by crystalline trypsin.While the rates of hydrolysis of α-benzoyl-l-argininamide and α-benzoyl-l-arginine isopropyl ester were unaffected by pressures of 8000 lb./in.², the hydrolysis of l-arginine methyl ester was slightly accelerated, the volume change of activation being ?6 ml.At 54.5 °C. and pH 8.1 high pressures accelerate the hydrolysis of α-benzoyl-l-argininamide by crystalline trypsin indicating that heat inactivation is retarded at higher pressures.     

Further studies of detergent-induced conformational transitions in proteins. Circular dichroism of ovalbumin, bacterial alpha-amylase, papain, and beta-lactoglobulin at various pH values.

The conformational transitions of ovalbumin, bacterial α-amylase, papain, and β-lactoglobulin were studied in the absence and presence of sodium dodecyl sulfate (SDS) between pH 2.75 and 12.0 by means of circular dichroism (CD) measurement. The weight ratios of SDS to protein in solutions were 14:1 in all experiments. The CD bands in the near-ultraviolet spectral region were strongly reduced by SDS, whereas those in the far-ultraviolet were enhanced. With the exception of the amylase, the mean residue ellipticities of the proteins at 222 nm were increased by SDS, especially in acidic solutions. At a pH of about 3.0, the [θ]₂₂₂ values approached ?17 (±2) · 10³ deg · cm² · dmol^?1. It is assumed that at a sufficiently low pH value the proteins which are complexed with SDS have a similar backbone conformation of moderate helical content. In alkaline solutions, the detergent effect was largely reduced due to electrostatic repulsion between the negatively charged protein and dodecyl ions. The near-ultraviolet spectra of ovalbumin, papain, and β-lactoglobulin at pH 6.4 were analyzed. Assignment of the resolved bands to the appropriate chromophores was also attempted.     

Protein hydration: A sucrose probe

The hydration of conalbumin, of myoglobin, of lysozyme, of carbon monoxide hemoglobin, of β-lactoglobin, of bovine serum albumin, of ovomucoid, of ribonuclease, and of egg albumin has been measured with equilibrium dialysis using sucrose as the probe at 30 °C. All proteins were at their isoelectric points except lysozymes and β-lactoglobulin and also samples of egg albumin which had been shifted to a more alkaline pH. Departure from their isoelectric points leads to an increase in the apparent protein hydration. Decreasing the temperature to 11.5 °C produces a slight increase in the hydration of egg albumin. A method is proposed for the calculation of protein hydration. The calculated protein hydration tends to be less than that determined experimentally for five of the proteins. There is satisfactory agreement with four of the proteins.     

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.