Human γS-Crystallin Mutation F10_Y11delinsLN in the First Greek Key Pair Destabilizes and Impairs Tight Packing Causing Cortical Lamellar Cataract.

Aromatic residues forming tyrosine corners within Greek key motifs are critical for the folding, stability, and order of ßγ-crystallins and thus lens transparency. To delineate how a double amino acid substitution in an N-terminal-domain tyrosine corner of the CRYGS mutant p.F10_Y11delinsLN causes juvenile autosomal dominant cortical lamellar cataracts, human γS-crystallin c-DNA was cloned into pET-20b (+) and a p.F10_Y11delinsLN mutant was generated via site-directed mutagenesis, overexpressed, and purified using ion-exchange and size-exclusion chromatography. Structure, stability, and aggregation properties in solution under thermal and chemical stress were determined using spectrofluorimetry and circular dichroism. In benign conditions, the p.F10_Y11delinsLN mutation does not affect the protein backbone but alters its tryptophan microenvironment slightly. The mutant is less stable to thermal and GuHCl-induced stress, undergoing a two-state transition with a midpoint of 60.4 °C (wild type 73.1 °C) under thermal stress and exhibiting a three-state transition with midpoints of 1.25 and 2.59 M GuHCl (wild type: two-state transition with Cm = 2.72 M GuHCl). The mutant self-aggregates upon heating at 60 °C, which is inhibited by α-crystallin and reducing agents. Thus, the F10_Y11delinsLN mutation in human γS-crystallin impairs the protein's tryptophan microenvironment, weakening its stability under thermal and chemical stress, resulting in self-aggregation, lens opacification, and cataract.

The Y46D Mutation Destabilizes Dense Packing of the Second Greek Key Pair of Human γC-Crystallin Causing Congenital Nuclear Cataracts.

The γ-crystallins are highly expressed structural lens proteins comprising four Greek key motifs arranged in two domains. Their globular structure and short-range spatial ordering are essential for lens transparency. Aromatic residues play a vital role in stabilizing Greek key folds by forming Greek key or non-Greek key pairs or tyrosine corners. We investigated the effects of the cataractogenic Y46D mutation in the second Greek key pair (Y46-Y51) of human γC-crystallin on its stability and aggregation. Wild-type and Y46D mutant human γC-crystallin were overexpressed in E. coli BL-21(DE3) PLysS cells, purified using ion-exchange and size-exclusion chromatography, and analyzed by fluorescence spectroscopy and circular dichroism spectroscopy. The Y46D mutation does not affect the γC-crystallin backbone conformation under benign conditions but alters the tryptophan microenvironment, exposing hydrophobic residues to the surface. The Y46D mutant undergoes a three-state transition under thermal stress with midpoints of 54.6 and 67.7 °C while the wild type shows a two-state transition with a midpoint of 77.6 °C. The Y46D mutant also shows a three-state transition under GuHCl stress with Cm values of 0.9 and 2.1 M while the wild type shows a two-state transition with a Cm of 2.4 M GuHCl. Mutant but not wild-type γC-crystallin forms light scattering particles upon heating at 65 °C. Overall, the Y46D CRYGS mutation leaves the protein fold intact under benign conditions but destabilizes the molecule by altering the tryptophan microenvironment and exposing hydrophobic residues to its surface, thus increasing its susceptibility to thermal and chemical stress with resultant self-aggregation, light scattering, and cataract.