Hydrophobic core mutations associated with cataract development in mice destabilize human gammaD-crystallin.
The human eye lens is composed of fiber cells packed with crystallins up to 450 mg/ml. Human gammaD-crystallin (HgammaD-Crys) is a monomeric, two-domain protein of the lens central nucleus. Both domains of this long lived protein have double Greek key beta-sheet folds with well packed hydrophobic cores. Three mutations resulting in amino acid substitutions in the gamma-crystallin buried cores (two in the N-terminal domain (N-td) and one in the C-terminal domain (C-td)) cause early onset cataract in mice, presumably an aggregated state of the mutant crystallins. It has not been possible to identify the aggregating precursor within lens tissues. To compare in vivo cataract-forming phenotypes with in vitro unfolding and aggregation of gamma-crystallins, mouse mutant substitutions were introduced into HgammaD-Crys. The mutant proteins L5S, V75D, and I90F were expressed and purified from Escherichia coli. WT HgammaD-Crys unfolds in vitro through a three-state pathway, exhibiting an intermediate with the N-td unfolded and the C-td native-like. L5S and V75D in the N-td also displayed three-state unfolding transitions, with the first transition, unfolding of the N-td, shifted to significantly lower denaturant concentrations. I90F destabilized the C-td, shifting the overall unfolding transition to lower denaturant concentrations. During thermal denaturation, the mutant proteins exhibited lowered thermal stability compared with WT. Kinetic unfolding experiments showed that the N-tds of L5S and V75D unfolded faster than WT. I90F was globally destabilized and unfolded more rapidly. These results support models of cataract formation in which generation of partially unfolded species are precursors to the aggregated cataractous states responsible for light scattering.
Moreau
Kate L
KL
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
King
Jonathan
J
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002386:Cataract / Q000235:genetics*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D038222:gamma-Crystallins / Q000737:chemistry* / Q000235:genetics*
C504903:CRYGD protein, human; D050505:Mutant Proteins; D011994:Recombinant Proteins; D038222:gamma-Crystallins