Colloidal Stability & Conformational Changes in β-Lactoglobulin: Unfolding to Self-Assembly

Abstract

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 rheological evolution with temperature. This study has, for the first time, demonstrated that this low frequency Raman data, in conjunction with the DSC endotherm, can be been utilized to deconvolve protein unfolding and aggregation/gelation. These findings can have important implications for the development of protein-based biotherapeutics, where the formulation viscosity, aggregation, and stability strongly affects efficacy or in foods where protein structuring is critical for functional and sensory performance.

A new methodology to assess the solubility of fatty acids: Impact of food emulsifiers

“…The volume size distribution of βlg (at 0.5 and 1.5% w/w) in SIF was determined in a previous work (Naso et al 2018). A monomodal particle size distribution was obtained, with a maximum value at 4.6 nm, which falls in between those corresponding to the monomeric (3.6 nm) and the dimeric form (6.9 nm) (Blake, Amin, Qi, Majumda, & Lewis, 2015).…”

Raman Spectroscopic Analysis of Highly-Concentrated Antibodies under the Acid-Treated Conditions

“…2 and 4), the decreased signals were detected upon the temperature increasing and thermal transition temperatures (T m ) and onset temperatures (T onset ) of three Raman bands of Phe, Trp, and amide I related to the secondary and tertiary structures were calculated (Tables I and II). The heating tests were also conducted in several studies using Raman spectroscopy and the decreased signals were observed with thermal stress and conformational changes of model proteins were discussed [12,13,15,16]. The T m derived from the respective bands showed almost similar values, indicating that the significant differences were not observed in these experiments.…”

“…Raman spectroscopy can provide conformational information derived from amino acid residues, especially aromatic ones and secondary structures as demonstrated by analyses of model proteins [10][11][12][13][14][15][16][17]. Spectral changes have been demonstrated to occur upon heating that result from alterations in protein conformation [12,13,15,16,18]. Our group carried out a study for high concentration solutions of an antibody [10].…”

Ionic strength and buffering capacity of emulsifying salts determine denaturation and gelation temperatures of whey proteins

“…Typical reported β-LG denaturation temperatures obtained via differential scanning calorimetry (DSC) at high protein concentrations and pH 6.5 to 7.0 range from 73.3 to 75.5°C (Damodaran, 1989;Verheul et al, 1998a;Haug et al, 2009). Higher temperatures are needed to denature β-LG at acidic pH (Haug et al, 2009;Blake et al, 2015).…”