Effect of crowding on several stages of protein aggregation in test systems in the presence of α-crystallin

Macromolecular crowding can facilitate protein-protein interactions in the cell, in particular aggregation processes. To characterize the anti-aggregation activity of chaperones under conditions mimicking the crowded environment in the cell, two basic test systems are used. Test systems of the first type are based on aggregation of target proteins undergoing unfolding under different factors. Dithithreitol-induced aggregation of alpha-lactalbumin is used as such a system. The increase in the duration of lag phase after the addition of the crowder (polyethylene glycol; PEG) to the system containing alpha-crystallin has been interpreted as a retardation of the stages that are the rate-limiting stages of the general process of aggregation (the nucleation stage and the stages of clusterization of nuclei). Test systems of the second type are based on aggregation of UV-irradiated proteins. Such test systems permit investigating the effects of different agents directly on the stages of aggregation of unfolded protein. UV-irradiated glycogen phosphorylase b (Phb) is used as a target protein. Analysis of the initial rate of aggregation after the addition of PEG at different points in time to the mixture of UV-irradiated Phb and alpha-crystallin allowed estimating the time of half-conversion for the structural rearrangement of the primary UV-irradiated Phb-alpha-crystallin complex. (C) 2015 Elsevier B.V. All rights reserved.