Electrostatic origin of in vitro aggregation of human γ-crystallin

The proteins alpha-, beta-, and gamma-crystallins are the major components of the lens in the human eye. Using dynamic light scattering method, we have performed in vitro investigations of protein-protein interactions in dilute solutions of human gamma-crystallin and alpha-crystallin. We find that gamma-crystallin spontaneously aggregates into finite-sized clusters in phosphate buffer solutions. There are two distinct populations of unaggregated and aggregated gamma-crystallins in these solutions. On the other hand, alpha-crystallin molecules are not aggregated into large clusters in solutions of alpha-crystallin alone. When alpha-crystallin and gamma-crystallin are mixed in phosphate buffer solutions, we demonstrate that the clusters of gamma-crystallin are prevented. By further investigating the roles of temperature, protein concentration, pH, salt concentration, and a reducing agent, we show that the aggregation of gamma-crystallin under our in vitro conditions arises from non-covalent electrostatic interactions. In addition, we show that aggregation of gamma-crystallin occurs under the dilute in vitro conditions even in the absence of oxidizing agents that can induce disulfide cross-links, long considered to be responsible for human cataracts. Aggregation of gamma-crystallin when maintained under reducing conditions suggests that oxidation does not contribute to the aggregation in dilute solutions. (C) 2013 AIP Publishing LLC.