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

Venkata Pulla Rao Vendra, Christian Ostrowski, Rebecca Clark, Marzena Dyba, Sergey G Tarasov, J Fielding Hejtmancik

Biochemistry 2023 Jun 20

filter terms:

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.

Citation

Venkata Pulla Rao Vendra, Christian Ostrowski, Rebecca Clark, Marzena Dyba, Sergey G Tarasov, J Fielding Hejtmancik. The Y46D Mutation Destabilizes Dense Packing of the Second Greek Key Pair of Human γC-Crystallin Causing Congenital Nuclear Cataracts. Biochemistry. 2023 Jun 20;62(12):1864-1877