Effect of the Air−Water Interface on the Stability of β-Lactoglobulin (original) (raw)
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Journal of Colloid and Interface Science, 2006
Unfolding of proteins has often been mentioned as an important factor during the adsorption process at air-water interfaces and in the increase of surface pressure at later stages of the adsorption process. This work focuses on the question whether the folding state of the adsorbed protein depends on the rate of adsorption to the interface, which can be controlled by bulk concentration. Therefore, the adsorption of proteins with varying structural stabilities at several protein concentrations was studied using ellipsometry and surface tensiometry. For β-lactoglobulin the adsorbed amount (Γ) needed to reach a certain surface pressure (Π) decreased with decreasing bulk concentration. Ovalbumin showed no such dependence. To verify whether this difference in behavior is caused by the difference in structural stability, similar experiments were performed with cytochrome c and a destabilized variant of this protein. Both proteins showed identical Π-Γ , and no dependence on bulk concentration. From this work it was concluded that unfolding will only take place if the kinetics of adsorption is similar or slower than the kinetics of unfolding. The latter depends on the activation energy of unfolding (which is in the order of 100-300 kJ/mol), rather than the free energy of unfolding (typically 10-50 kJ/mol).
The impact of high hydrostatic pressure on structure and dynamics of β-lactoglobulin
Biochimica et biophysica acta, 2013
Methods: Combining small-angle X-ray and neutron scattering measurements with inelastic neutron scattering experiments, we investigated the impact of high hydrostatic pressure on the structure and dynamics of β-lactoglobulin (βLG) in aqueous solution. Background: βLG is a relatively small protein, which is predominantly dimeric in physiological conditions, but dissociates to monomer below about pH 3. Results: High-pressure structural results show that the dimer-monomer equilibrium, as well as the protein-protein interactions, are only slightly perturbed by pressure, and βLG unfolding is observed above a threshold value of 3000 bar. In the same range of pressure, dynamical results put in evidence a slowing down of the protein dynamics in the picosecond timescale and a loss of rigidity of the βLG structure. This dynamical behavior can be related to the onset of unfolding processes, probably promoted from water penetration in the hydrophobic cavity. General significance: Results suggest that density and compressibility of water molecules in contact with the protein are key parameters to regulate the protein flexibility.
Unfolding and refolding of bovine β-lactoglobulin monitored by hydrogen exchange measurements
Journal of Molecular Biology, 1999
Bovine b-lactoglobulin (b-LG) is a widely studied protein belonging to the lipocalin family, whose structural characterisation has been reported by X-ray crystallography and NMR studies at physiological and acidic pH, respectively. Bovine b-LG consists of nine antiparallel b-sheets and a terminal a-helix segment. The b-sheets form a calyx structure with a hydrophobic buried cluster conferring stability to the protein while a hydrophobic surface patch provides stabilising interactions between the barrel and the¯anking terminal helix.
Biophysical Journal, 2007
The b/a transition of b-lactoglobulin, a globular protein abundant in the milk of several mammals, is investigated in this work. This transition, induced by the cationic surfactant dodecyltrimethylammonium chloride (DTAC), is accompanied by partial unfolding of the protein. In this work, unfolding of bovine b-lactoglobulin in DTAC is compared with its unfolding induced by the chemical denaturant guanidine hydrochloride (GnHCl). The final protein states attained in the two media have quite different secondary structure: in DTAC the a-helical content increases, leading to the so-called a-state; in GnHCl the amount of ordered secondary-structure decreases, resulting in a random coil-rich final state (denatured, or D, state). To obtain information on both mechanistic routes, in DTAC and GnHCl, and to characterize intermediates, the kinetics of unfolding were investigated in the two media. Equilibrium and kinetic data show the partial accumulation of an on-pathway intermediate in each unfolding route: in DTAC, an intermediate (I 1 ) with mostly native secondary structure but loose tertiary structure appears between the native (b) and a-states; in GnHCl, another intermediate (I 2 ) appears between states b and D. Kinetic rate constants follow a linear Chevron-plot representation in GnHCl, but show a more complex mechanism in DTAC, which acts like a stronger binding species.
Denaturation and aggregation of β-lactoglobulin—a preliminary molecular dynamics study
Food Hydrocolloids, 2007
The heat-induced denaturation and the molecular basis for aggregation in b-lactoglobulin has been investigated using a combination of molecular dynamics simulation and bioinformatics analysis. Molecular dynamics has been used to simulate the temperature induced unfolding of a single b-lactoglobulin molecule. Although the study is carried out at an elevated temperature to speed up the simulation, it confirms the experimental observation that the b-sheet structure in the protein is more stable to heat than the a-helical regions. We have also used bioinformatics analysis to search the b-lactoglobulin primary sequence for potential minimal sequences that may act as initiators for fibril formation in fine-stranded gels. Two potential candidate sequences were identified, and one GDLEIL was shown by molecular dynamics simulation to be able to form anti-parallel b-sheet with copies of itself. The potential significance of the minimal sequences to fine-stranded gel formation is discussed by way of analogy with the postulated mechanisms for amyloid fibril formation. r
Surface-Induced Unfolding of Human Lactoferrin
Langmuir, 2005
We have determined the structural conformations of human lactoferrin adsorbed at the air/water interface by neutron reflectivity (NR) and its solution structure by small angle neutron scattering (SANS). The neutron reflectivity measurements revealed a strong structural unfolding of the molecule when adsorbed at the interface from a pH 7 phosphate buffer solution (PBS with a total ionic strength at 4.5 mM) over a wide concentration range. Two distinct regions, a top dense layer of 15-20 Å on the air side and a bottom diffuse layer of some 50 Å into the aqueous subphase, characterized the unfolded interfacial layer. At a concentration around 1 g dm -3 , close to the physiological concentration of lactoferrin in biological fluids, the adsorbed amount was 5.5 × 10 -8 mol m -2 in the absence of NaCl, but the addition of 0.3 M NaCl reduced protein adsorption to 3.5 × 10 -8 mol m -2 . Although the polypeptide distributions at the interface remained similar, quantitative analysis showed that the addition of NaCl reduced the layer thickness. Parallel measurements of lactoferrin adsorption in D 2O instead of null reflecting water confirmed the unfolded structure at the interface. Furthermore, the D2O data indicated that the polypeptide in the top layer was predominantly protruded out of water, consistent with it being hydrophobic. In contrast, the scattering intensity profiles from SANS were well described by a cylindrical model with a diameter of 47 Å and a length of 105 Å in the presence of 0.3 M NaCl, indicating a retention of the globular framework in the bulk solution. In the absence of NaCl but with the same amount of phosphate buffer, the length of the cylinder increased to some 190 Å and the diameter remained constant. The length increase is indicative of changes in distance and orientation between the bilobal monomers due to the change in charge interactions. The results thus demonstrate that the surface structural unfolding was caused by the exposure of the protein molecule to the unsymmetrical energetic balance following surface adsorption.
Colloidal Stability & Conformational Changes in β-Lactoglobulin: Unfolding to Self-Assembly
International journal of molecular sciences, 2015
A detailed understanding of the mechanism of unfolding, aggregation, and associated rheological changes is developed in this study for β-Lactoglobulin at different pH values through concomitant measurements utilizing dynamic light scattering (DLS), optical microrheology, Raman spectroscopy, and differential scanning calorimetry (DSC). The diffusion interaction parameter kD emerges as an accurate predictor of colloidal stability for this protein consistent with observed aggregation trends and rheology. Drastic aggregation and gelation were observed at pH 5.5. Under this condition, the protein's secondary and tertiary structures changed simultaneously. At higher pH (7.0 and 8.5), oligomerizaton with no gel formation occurred. For these solutions, tertiary structure and secondary structure transitions were sequential. The low frequency Raman data, which is a good indicator of hydrogen bonding and structuring in water, has been shown to exhibit a strong correlation with the rheologi...
Chemical Physics Letters, 2006
Small-angle neutron scattering (SANS) of solutions of glucose/xylose isomerase from Streptomyces rubiginosus was measured as a function of pressure. It is shown that the structure of the enzyme in solution as seen by SANS is practically the same as that in the crystal and does not change with pressure up to 150 MPa. This reflects the unusually high structural stability of this material, which makes it extremely interesting to use as a secondary standard for pressure-dependent SANS experiments. This lack of pressure dependence of the SANS data also indicates that any possible change in hydration of the protein induced by pressure is not visible in the SANS curves. An appropriate correction procedure must be used for the SANS data in order to account for the distortion of the intensity curve due to hard-sphere and electrostatic interactions. After this correction, the isomerase can be readily used as a secondary standard for SANS measurements. research papers J. Appl. Cryst. (2009). 42, 461-468 Ewa Banachowicz et al. Glucose isomerase conformation in solution 463
Adsorption of beta-Lactoglobulin variants A and B to the air-water interface
International Journal of Food Science and Technology, 1999
The adsorption of proteins to interfaces is a vital and complex process for the formation and stabilization of multiphase food systems (emulsions and foams). The process of protein adsorption is generally understood only at the phenomenological level, as the complexity of protein unfolding during adsorption is very difficult to predict and model. By comparing proteins with very similar structures, it is possible to attribute observed changes in adsorption behaviour. The A and B genetic variants of -lactoglobulin (-lg) differ by only two amino acids (Asp-64, Val-118 in A, and Gly-64, Ala-118 in B), thus making them ideal candidates for this type of comparison. In this study we monitored the surface behaviour of -lg A and B, measuring the surface tension and surface dilational modulus of adsorbed protein, and the compression behaviour of spread protein films. At pH 7, variant B lowered the surface tension and increased the surface dilational modulus more rapidly than variant A. Raising the pH to 7.8 should increase the level of dissociation into monomers. Indeed, this was confirmed by the rate of adsorption, which increased in both cases. Also, the surface tension of both variants was much lower than at pH7. Variant B was less sensitive to the change of pH than A. Regardless of pH, after 3 h adsorption the difference between the variants in surface tension or surface dilational modulus was negligible. The differences in surface behaviour between the variants are discussed in terms of interactions between monomers at and with the interface, and the dimer : monomer equilibrium in the bulk solution.