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Properties of Spray-freeze-dried Products and their Characterization
Published in S. Padma Ishwarya, Spray-Freeze-Drying of Foods and Bioproducts, 2022
Apart from the chromatography techniques, the aggregation behavior of spray-freeze-dried proteins has also been ascertained by subjecting the rehydrated SFD powder to gel electrophoresis (Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis, also known as SDS-PAGE) and ultraviolet (UV) spectrophotometry. With gel electrophoresis, the presence of an intense monomer band, dimer band and some aggregates in the SDS gel lanes signifies the occurrence of protein aggregation during freezing (Figure 10.5). The dimers and minimal aggregates in rehydrated SFD are the results of protein inactivation caused by its partial and irreversible unfolding to an inactive molten globule state. The above inactivation events occur during the atomization stage, wherein the protein adsorbs and unfolds at the large liquid/air interface of the spray droplets. These unfolded proteins do not refold on rehydration and therefore continues to be inactive, but are not aggregated. Thus, studies have ascertained that protein aggregation is minimal during SFD/rehydration (Sonner, Maa & Lee, 2002).
The characteristics of molten globule states and folding pathways strongly depend on the sequence of a protein
Published in Molecular Physics, 2018
M.J.J. Dijkstra, W.J. Fokkink, J. Heringa, E. van Dijk, S. Abeln
While the native protein fold is highly specific, more dynamic, relatively compact states have also been observed for many real proteins; such states are usually referred to as ‘molten globules’ [13,14]. These molten globules are much more compact than the fully unfolded coil state, but much less specific than the folded state; intra-chain contacts between the residues of the protein form a fluctuating ensemble [14–17]. Over the last decade, it has become increasingly clear that there is a very diverse spectrum of molten globule-like states, ranging from near native compact structures (‘dry’ molten globules) to much more dynamic and more solvent accessible structures (‘wet’ molten globules) [18–20]. These state ensembles also vary greatly in the extent to which their cores are solvent accessible, spanning several orders of magnitude [19]. Experimentally, the denaturation of molten globule states coincides with a peak in the heat capacity, although such peaks are much smaller than those observed for unfolding [21] the native structure. Note that some proteins have even been reported to be able to function in a molten globule-like state [19,22].
Quinoline yellow (food additive) induced conformational changes in lysozyme: a spectroscopic, docking and simulation studies of dye-protein interactions
Published in Preparative Biochemistry & Biotechnology, 2020
Mohd Shahnawaz Khan, Sheraz Bhatt, Shams Tabrez, Md Tabish Rehman, Majed Saleh Alokail, Mohamed F. AlAjmi
ANS is a small organic compound, used to probe the surface hydrophobicity in proteins, and fluoresce only upon binding with hydrophobic materials. It is also widely used for the characterization and identification of folding intermediate states such as molten globule, pre molten globule as well as partially folded states of proteins. As depicted in Fig. 3, HEWL upon incubation with QY showed a continuous decline in ANS fluorescence signal, the extent of which varied as a function of QY concentration. This ANS fluorescence quenching may be due to the cross-linking of lysozyme molecules leading to burial of some portions of hydrophobic patches that were otherwise exposed in native form of the enzyme. Moreover, at 5 µM and 10 µM, the HEWL showed considerable residual ANS signal suggesting the formation of a partially unfolded state or pre-molten globule state. This may be because ligand interaction buries some hydrophobic patches of the protein in the core that otherwise remain exposed reflecting the structural alteration as mentioned earlier. However, at higher concentrations of QY (25–100 µM), HEWL showed negligible ANS signal which may be due to crosslinking and coalescing of enzyme molecules in such a way that surface hydrophobicity remains elusive. Thus, the ANS results suggest unfolding and other structural alterations of HEWL upon incubation with food additive dye QY, giving rise to some structural intermediates at lower doses, which upon incubation with higher concentrations of QY undergo cross-linking and coalescing to produce aggregate species. Unfolding and conformational changes in proteins leading to aggregate and amyloid formational have been reported earlier too.[7,18]