Infantile cataracts constitute one of the most important causes of childhood blindness worldwide. Human gamma S-crystallin is the most abundant protein in the eye lens cortex. A missense mutant of human gamma S-crystallin, Y67N (abbreviated hereafter as gamma S-Y67N) is recently reported to be associated with dominant infantile cataracts. To understand the structural basis for gamma S-Y67N to cause lens opacification, we constructed, expressed and purified gamma S-Y67N and its wild-type (abbreviated hereafter as gamma S-WT) and studied the structural and functional differences between them in solution using circular dichroism (CD), differential scanning calorimetry (DSC), fluorescence spectroscopy and extrinsic spectral probes. Extensive equilibrium characterization indicate that replacement of the highly conserved Tyr at 67th position by Asn distorts the conserved Tyr corner at the second Greek key motif in the N-terminal domain (NTD) and leads to substantial loss of structural stability. Our intrinsic fluorescence quenching results reveal differential in-vitro refolding kinetics identifying partially folded kinetic intermediates for both proteins. Extrinsic fluorescence studies further reveal loosening up of the compact structure of gamma S-crystallin upon mutation associated with enhanced aggregation. As Ca2+ homeostasis is a crucial regulator of lens transparency, we further investigated the Ca2+-binding properties of gamma S-WT and gamma S-Y67N by isothermal titration calorimetry (ITC) to identify lens Ca2+ distribution in health and in disease. Overall, our results highlight the vital role of conserved Tyr corners in stabilizing Greek key motifs and provide useful structural and functional insights into the mechanism of cataract formation in humans. (C) 2018 Elsevier Inc. All rights reserved.