Colloidal Stability & Conformational Changes in β-Lactoglobulin: Unfolding to Self-Assembly (original) (raw)
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Heat-Induced Gelation of β -Lactoglobulin. 1. Time-Resolved Dynamic Light Scattering
Macromolecules, 2000
The time evolution of the dynamics of globular protein during the gelation process via aggregation has been studied by time-resolved dynamic light scattering (DLS) for-lactoglobulin (-LG) in aqueous solutions at pH 2 and 7. The following facts were disclosed: (1) The scattered intensity, 〈I〉T, started to fluctuate, and the intensity-time correlation function (ITCF) exhibited a power-law behavior (g (2) (τ)-1 ∼ τ R-1) at the gelation threshold, tth. (2) The variations of 〈I〉T's were different between the two pHs, a gradual increase for pH 2 and a stepwise increase followed by a plateau for pH 7. (3) The exponent R was found to be strongly dependent on pH. The values of R were 0.51 (0.05 for pH 2 and 0.74 (0.05 for pH 7. (4) A strong concentration and pH dependence of tth was also observed in both cases. These findings indicate that-LG gels formed at pH 2 and pH 7 have different architectures, i.e., loosely tied networks (for pH 2) and fractal aggregates (for pH 7) of protein molecules.
Complexation of β-Lactoglobulin Fibrils and Sulfated Polysaccharides
Biomacromolecules, 2011
We have investigated the thermodynamic and dynamic behavior of multistranded β-lactoglobulin protein fibrils in water, by combining static, dynamic, and depolarized dynamic light scattering (SLS, DLS, DDLS), small angle neutron scattering (SANS), rheology, and cryogenic transmission electron microscopy (cryo-TEM). We focus on the region of the phase diagram at which ionic strength and concentration changes induce transitions in gelation and lyotropic liquid crystalline behavior. An increase in ionic strength, induced by NaCl salt, progressively causes the phase transitions from nematic (N) to gel (G) phases; a further increase causes the transition to a translucent phase and to a macroscopic phase separation, respectively. An increase in fibril concentration induces first a phase transition from an isotropic (I) to a nematic phase (N); a further increase induces the formation of a gel phase. The protein gel strength is investigated by rheology measurements. SANS and osmotic compressibility calculated by SLS measurements clearly capture the main features of the IN transition of β-lactoglobulin protein fibrils. The form and structure factors measured by scattering experiments are analyzed by the polymer reference interaction site model (PRISM). Dynamics of the protein fibrils at different concentrations, measured by polarized and depolarized dynamic light scattering, show both individual and collective diffusion after the isotropic−nematic transition. Above this transition, cryo-TEM images further demonstrate the alignment of the protein fibrils, which is quantified by a 2D order parameter. This work discusses comprehensively, both experimentally and theoretically, the thermodynamics and dynamic features of β-lactoglobulin amyloid fibrils in a vast region of the concentration−ionic strength phase diagram.
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
Polymer, 2008
The gelation mechanism of b-lactoglobulin (bLG) aqueous solutions was investigated by dynamic light scattering (DLS) and small-angle neutron scattering (SANS). Temperature-and pressure-jump experiments, respectively, abbreviated as T-jump (from 20 to 75 C; T-jump) and P-jump (from 0.1 to 315 MPa) were carried out and the time evolution of gel structure was monitored by DLS and SANS as a function of time. The gelation threshold was determined by DLS as the point when nonergodicity appeared. In the case of T-jump, a rapid increase of the time-average scattered intensity, CID T , and a steep decrease of the initial amplitude of the intensity-intensity time correlation function, s 2 I , were observed at the gelation threshold. On the other hand, P-jump showed a gradual increase of the CID T and a continuous decrease of the s 2 I. It was revealed by SANS that bLG underwent thermal denaturation, resulting in a formation of gels consisting of densely aggregated unfolded bLG oligomers. On the other hand, the pressure-induced gels were found to be a fractal aggregates consisting of primary particles of bLG monomers. The difference in the gel structure as well as gelation mechanism between bLGs treated by T-jump and P-jump is discussed in comparison with T-induced and P-induced microphase separation of amphiphilic block copolymers in water [Osaka N, Shibayama M.
Food Hydrocolloids, 2005
Using confocal laser scanning microscopy, we have followed in time the effect of a non-ionic surfactant Tween 20 on the evolution of the microstructure during the thermal gelation of b-lactoglobulin (b-lg). At pH 6.8, the final gel is fine-stranded, and addition of Tween 20 had no discernible effect on the final microscopic gel structure. However, for surfactant/protein molar ratios between RZ0.5 and 2, small aggregates were observed to appear in the protein solution, and they disappeared again upon further heating towards the gel point. This aggregation process was found to be completely reversible if the gel point was not reached. The size and concentration of the aggregates depended on R and on the heating rate. At pH 5.5 with 0.05 M NaCl present, a preliminary study indicated the formation of a coarse b-lg gel on heating, and the degree of inhomogeneity was observed to be greater in the presence of surfactant. These findings demonstrate the potential of confocal microscopy for studying the effect of protein-surfactant interactions on the evolving structure of aggregates during the heat-induced gelation of globular food proteins.
Growth and structure of aggregates of heat-denatured beta-Lactoglobulin
International Journal of Food Science and Technology, 1999
The aggregation of the globular protein -lactoglobulin after heat-denaturation was studied in aqueous solution at pH 7 using static and dynamic light scattering. The structure of the aggregates is self-similar with fractal dimension 2.0. Size exclusion chromatography and dynamic light scattering measurements show that the aggregates have a broad size distribution. Initially clusters of about 85 proteins and 15 nm radius are formed which are the elementary units of the larger fractal aggregates. At low ionic strength the formation of the larger aggregates is impeded by electrostatic interactions.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Highlights • Under the action of GnHCl monomerization followed by denaturation of monomers take place. • At around ~ 308 K formation of monomers has been noticed. • Upto 338 K reversible formation of small aggregates (diameter ~ 77 Å) takes place. • Above 338 K irreversible formation of larger aggregates (diameter ~ 117 Å) is observed. Abstract: β-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of β-lactoglobulin at pH 7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of β-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of β-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~ 55 Å, ~ 29 Å and ~ 37 Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57 kcal mol− 1 and 8.93 kcal mol− 1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of β-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~ 77 Å and ~ 117 Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69 kcal mol− 1 and 9.09 kcal mol− 1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.
Journal of Raman Spectroscopy, 2012
Raman spectroscopy investigations carried out simultaneously in the low wavenumber range (10-400 cm −1 ) and in the amide I region (1500-1800 cm −1 ). These investigations show common features between the denaturation processes at low and high temperatures. The denatured states are reached via an intermediate state characterized by a soft tertiary structure without detectable conformational changes. This intermediate is intimately connected with a tetrahedral hydrogen-bond structure of water which extends over a limited range. It is shown that the disruption of the hydrogen-bond network of D 2 O has an important consequence on the solvent dynamics, which controls protein dynamics and is characterized by an anharmonic behavior. By monitoring the amide I mode, conformational changes are detected at low temperature (below 5 • C) and determined to be similar to those detected at high temperature in the presence of urea near 65 • C, and in the absence of urea near 80 • C. The conformational changes are described as a loss of α-helix structures and a concomitant formation of β-sheets. The temperature dependence of the amide I wavenumber in BLG dissolved in the 4 M urea aqueous solution was interpreted on the basis of a two-state model, leading to the protein stability curve related to its molecular conformation.
Food Hydrocolloids, 1996
Association properties of f3-lactoglobulin AB (f3-Lg) fractionated by gel permeation chromatography (GPC) was studied using dynamic light scattering (DLS) at a concentration of 5% w/vand pH 7.0 from 25 to 70°e. f3-Lg fraction with a molecular weight of 18.4 kDa by GPC (monomeric) showed self-association at 25°e. At 25°C~58% of the protein had an apparent mean diameter <10 nm and the rest between 10and 100 nm. On heating to 35°C all the protein existed as monomers and dimers. With further heating association increased; large particles with an apparent size of100-599 nm were seen >45°C indicating that some degree of denaturation occurs even at 45°e. The amount of aggregate <10 nm in size decreased sharply >65°e. Data indicate that f3-Lg that was monomeric during GPC, where a linear velocity gradient may exist, was associated even at 25°C under quiescent conditions of the DLS experiments. Association increased progressively with temperature and denaturation, as observed by the formation of large aggregation, starting at 45°e.