Mechanism of aggregation of UV-irradiated glycogen phosphorylase b at a low temperature in the presence of crowders and trimethylamine N-oxide

摘要

Abstract To characterize the initial stages of protein aggregation, the kinetics of aggregation of UV-irradiated glycogen phosphorylase b (UV-Ph b ) was studied under conditions when the aggregation proceeded at a low rate (10°C, 0.03M Hepes buffer, pH6.8, containing 0.1M NaCl). Aggregation of UV-Ph b was induced by polyethylene glycol and Ficoll-70, acting as crowders, or a natural osmolyte trimethylamine N-oxide (TMAO). It has been shown that the initial rate of the stage of aggregate growth is proportional to the protein concentration squared, suggesting that the order of aggregation with respect to the protein is equal to two. It has been concluded that the aggregation mechanism of UV-Ph b at 10°C in the presence of crowders includes the nucleation stage and stages of protein aggregate growth (the basic aggregation pathway). The aggregation mechanism is complicated in the presence of TMAO, and the stage of aggregate-aggregate assembly induced by TMAO should be added to the basic aggregation pathway. It has been shown that the ability of TMAO at a low concentration (0.05M) to induce aggregation of UV-Ph b is due to the decrease in the absolute value of zeta potential of the protein in the presence of TMAO. Graphical abstract Display Omitted Highlights ? Kinetic regime of aggregation of UV-irradiated glycogen phosphorylase b at 10°C has been established. ? Crowders (Ficoll-70 and polyethylene glycol) and osmolyte trimethylamine N-oxide (TMAO) stimulate protein aggregation. ? The initial rate of the stage of aggregate growth is proportional to the protein concentration squared. ? In contrast to crowders, TMAO induces sticking of the aggregates formed in the aggregation process.