The Nonequilibrium Phase and Glass Transition Behavior of β-Lactoglobulin (original) (raw)
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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...
Heat-and cold-setting gels of β-lactoglobulin solutions. A DSC and TEM study
We extracted data from the differential scanning calorimetric signals obtained with two different solutions of 13lactoglobulin at pH 3.5 and 7 and at a concentration which favours protein-protein interactions. The transition peaks were partially reversible only with solutions at pH 3.5. The temperatures corresponding to the first increase in heat flow, Oonset, and to the amplitude of maximum deviation, Omax, and the calorimetric enthalpy changes of heat reaction, ArH(cal) were higher at pH 3.5 than at pH 7. These differences indicated higher conformational stability at pH 3.5 than at pH 7" the heat transitions from the initial to the final conforrnational states could process through the reversible 'two-state model' at pH 3.5, while stable intermediate species might be created at pH 7. Isothermal treatments at Oonset<O<Omax+5 K, applied to the study of the solgel transition showed that solutions which denatured reversibly formed cold-setting gels, while solutions which denatured irreversibly formed heat-setting gels. Furthermore, the curve (O-t) which demarcated the diagrams of the sol-gel states intersected at O approx, equal to Omax and the gel structures observed by transmission electronic microscope showed that cold-setting gels were constituted by linear aggregates ('string of beads') while heat-setting gels were constituted by very large clumped aggregates. All these results are discussed in terms of protein-protein interaction forces.
Journal of Agricultural and Food Chemistry, 1995
The main purpose of this study was to determine the extent to which the process of foaming changes the structure ob P-lactoglobulin (p-Lg). ,B-Lg unfolded while existing as a foam, but changes in conformation were reversible upon collapse of the foam. A plot of the overrun (a measure of air incorporation in the foam) for p-Lg foams (pH 7) versus temperature (3-45 "C) was a sigmoidal curve that resembled a two-state denaturation curve. Foaming properties were influenced by temperature and pH, which was attributed to differences in protein structure, in diffusion rate and in the number of molecules in solution for p-Lg at pH 9 as compared to pH 7. There was a strong correlation (r > 0.9) between the secondary structure of p-Lg in solution and the observed foaming properties. A direct link between the structure of p-Lg in solution and its foaming properties was established.
Relating foam and interfacial rheological properties of β-lactoglobulin solutions
Soft matter, 2014
We have determined bulk rheology of β-lactoglobulin (BLG) foams and surface viscoelasticity of corresponding protein solutions by varying pH as well as type, valency and concentration of the added salt in a wide range. Foam rheology was characterized by the storage modulus G0, the apparent yield stress τy, and the critical strain γc,foam defining the cessation of the linear viscoelastic response. These quantities were determined at gas volume fractions ϕ between 82% and 96%. Surface viscoelasticity was characterized in shear and dilation, corresponding shear and dilational moduli G, E' as well as the critical stress τc,surface and strain γc,surface marking the onset of non-linear response in oscillatory surface shear experiments were determined at fixed frequency. Beyond the widely accepted assumption that G0 and τy are solely determined by the Laplace pressure within the droplets and the gas volume fraction we have found that both quantities strongly depend on corresponding int...
The Glass Transition Behavior of the Globular Protein Bovine Serum Albumin
Biophysical Journal, 2003
The glass-like transition behavior of concentrated aqueous solutions of bovine serum albumin was examined using rheological techniques. At mass fractions [0.4, there was a marked concentration dependence of viscosity with a glasslike kinetic arrest observed at mass fractions in the region of 0.55. At mass fractions [0.6 the material behaved as a solid with a Young's modulus rising from ;20 MPa at a mass fraction of 0.62-1.1 GPa at 0.86. The solid was viscoelastic and exhibited stress relaxation with relaxation times increasing from 33 to 610 s over the same concentration range. The concentration dependence of the osmotic pressure was measured, at intermediate concentrations, using an osmotic stress technique and could be described using a hard sphere model, indicating that the intermolecular interactions were predominantly repulsive. In summary, a major structural relaxation results from the collective motion of the globules at the supra-globule length scale and, at 208C, this is arrested at water contents of 40% w/w. This appears to be analogous to the glass transition in colloidal hard spheres.
Effect of the Air−Water Interface on the Stability of β-Lactoglobulin
The Journal of Physical Chemistry B, 2007
We report the X-ray and neutron reflectometry measurements of the structural changes caused by chemical denaturation of a surface excess of the bovine milk protein, -lactoglobulin. The thickness of the diffuse protein surface layer was used as an order parameter as there was no corresponding increase in the surface excess as a function of guanidinium chloride (G.HCl) concentration. A thermodynamic analysis performed gave the interfacial free energy of unfolding in the absence of a denaturant (∆G 0 ). This energy, lower than the free energy of unfolding bulk solution, shows that the air-water interface has a destabilizing effect on protein structure up to 50 kJ mol -1 .
Fibrillar β-Lactoglobulin Gels: Part 2. Dynamic Mechanical Characterization of Heat-Set Systems
Biomacromolecules, 2004
Oscillatory shear rheometry (mechanical spectroscopy) has been used to study the heat-set gelation of -lactoglobulin at pH 2. Modulus-concentration relationships were obtained by extrapolating cure data to infinite time. In terms of theory, these fail to provide a clear distinction between the fractal description of biopolymer gels and the classical random f-functional polycondensation branching theory (cascade) approach, though the latter is preferred. Critical exponents for the sol-gel transition, derived from these data, are also discussed. Where gel time-concentration results are concerned the fractal model makes no predictions, and the cascade approach in its simplest form must be rejected in favor of a more sophisticated version involving delivery of fibrils by nucleation and growth into the random aggregation process. Over the limited concentration range accessed experimentally, cure data for the different -lactoglobulin solutions, reduced to the universal form G′/G′ inf versus t/t gel , superimposed well for samples heated both at 80 and 75°C and for different batches of protein. Studies of the frequency responses of the fully cured gels confirm the validity of the gel description given to these materials, and a study of the temperature dependence of the frequency spectrum suggests a fall in the elastic component of the modulus as temperature decreases. This contrasts with what has been found for other heat-set globular protein gels such as those from serum albumin where the gel modulus increases at lower temperatures. The present results are in good agreement with more limited amounts of pH 2 -lactoglobulin data published earlier, though some differences arise through a previous neglect of measurement "dead time".
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.