Human
gamma-crystallins are long-lived, unusually stable
proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone
alpha-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset
cataract is believed to represent an aggregated state of partially unfolded and covalently damaged
crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of
cataract proteins have made it difficult to identify the conformation of the human
gamma-crystallin substrate species recognized by human
alpha-crystallin. The three major human lens monomeric
gamma-crystallins, gammaD, gammaC, and gammaS, all refold in vitro in the absence of chaperones, on dilution from denaturant into
buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human alphaB-
crystallin chaperone during refolding suppressed the aggregation pathways of the three human
gamma-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The alphaB-
crystallin oligomers formed long-lived stable complexes with their gammaD-
crystallin substrates. Using
alpha-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone-substrate complex. Binding of substrate
gamma-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/
protein aggregation models for
cataract, with
alpha-crystallin suppressing aggregation of damaged or unfolded
proteins through early adulthood but becoming saturated with advancing age.