Biophysical Elucidation of Fibrillation Inhibition by Sugar Osmolytes in α-Lactalbumin: Multispectroscopic and Molecular Docking Approaches

Abstract

Protein aggregation is among the most challenging new frontiers in protein chemistry as well as in molecular medicine and has direct implications in protein misfolding. This study investigated the role of sugar molecules (glucose, fructose, sucrose, and the mixture of glucose and fructose) in protecting the structural integrity of α-lactalbumin (α-LA) against aggregation. The research focused here is the inhibitory capabilities of sugars against α-LA fibril formation investigated employing diverse multispectroscopic and microscopic techniques. The aggregation was induced in α-LA thermally with a change in concentration. UV–vis spectroscopy, ThT binding assay, Trp fluorescence, Rayleigh scattering, and turbidity assay depicted synchronized results. Further, transmission electron microscopy (TEM) complemented that a mixture of glucose and fructose was the best inhibitor of α-LA fibril formation. Inhibition of α-LA aggregation by sugar osmolytes is attributed to the formation of hydrogen bonds between these osmolytes, as evidenced by the molecular docking results. This hydrogen bonding is a key player that prevents aggregation in α-LA in the presence of sugar osmolytes. This study provides an insight into the ability of naturally occurring sugar osmolytes to inhibit fibril formation and can serve as a platform to treat protein misfolding and aggregation-oriented disorders.

Heparin Accelerates the Protein Aggregation via the Downhill Polymerization Mechanism: Multi-Spectroscopic Studies to Delineate the Implications on Proteinopathies

“…The formation of BSA aggregates with heparin was monitored through the ANS fluorescence emission measurements using the Jasco FP-6200 spectrofluorometer at 25 °C using a 1 cm cuvette, with both emission and excitation slit widths fixed at 10 nm, a data pitch of 1 nm, and a scanning speed of 125 nm min –1 . 89 Experiments of ANS binding were done at the excitation wavelength of 360 nm. The molar ratio of ANS to protein BSA was kept as 20:1 (100 μM ANS/5 μM BSA) for all the ANS binding experiments in 25 mM phosphate buffer at pH 7.0.…”

Trehalose Restrains the Fibril Load towards α-Lactalbumin Aggregation and Halts Fibrillation in a Concentration-Dependent Manner

“…To evaluate the aggregation profile of the apo form of α-LA, thermal transition curves at a fixed concentration of the protein (1.5 mg mL −1 ) as used earlier [ 27 ] were measured in the presence of different molar concentrations of trehalose (0.5, 0.75 and 1.0 M) by following changes in absorbance at 400 nm ( A 400 ) in the temperature range 20–85 °C at pH 4.5. Figure 1 A shows the results of these measurements.…”

“…The aggregation was induced by heating the protein solution at 70 °C for 30 min. This heated sample was cooled down to 25 °C to measure turbidity at 350 nm by using a UV-Vis spectrophotometer (Jasco UV-660) as described earlier [ 27 ].The turbidity measurement was also made using a proper blank of a native apo-α-LA in a buffer (i.e., in the absence of trehalose).…”

“…The samples were cooled down to 25 °C, and fluorescence spectra measurements were taken in a Jasco FP-6200 spectrofluorometer using a 1 cm quartz cell. During these measurements, both the excitation and emission slit width were kept at 10 nm, and a 1 cm pathlength cell was used as described earlier [ 27 ]. All solutions contained 1.5 mg mL −1 protein and 15 mg mL −1 ThT [ 27 ].…”

“…In the current research, we explored the effect of trehalose on α-LA aggregation. This study was motivated by our earlier work that showed saccharides as potential osmolytes interceded with the aggregation of several proteins [ 27 ]. Proteins are stabilized in the presence of trehalose in the native state and help rejuvenate denatured proteins by maintaining steady non-native states [ 12 , 28 ].…”