GammaS-crystallin, important in maintaining lens transparency, is a monomeric betagamma-crystallin comprising two paired homologous domains, each with two Greek key motifs. An autosomal dominant cortical progressive cataract has been associated with a G18V mutation in human gammaS-crystallin. To investigate the molecular mechanism of this cataract and confirm the causative nature of the G18V mutation, we examined resultant changes in conformation and stability. Human gammaS-crystallin cDNA was cloned into pET-20b(+), and the G18V mutant was generated by site-directed mutagenesis. Recombinant HgammaS-crystallins were expressed in Escherichia coli and purified by ion-exchange and size-exclusion chromatography. By analytical ultracentrifugation wild-type and mutant HgammaS-crystallins are monomers of about 21.95 +/- 0.21 and 20.89 +/- 0.18 kDa, respectively, and have similar secondary structures by far-UV CD. In increasing levels of guanidine hydrochloride (GuHCl), a sharp red shift in fluorescence lambda(max) and increase in emission correlating with exposure of tryptophans to the protein surface are detected earlier in the mutant protein. Under thermal stress, the G18V mutant begins to show changes in tryptophan fluorescence above 42 degrees C and shows a Tm of 65 degrees C as monitored by CD at 218 nm, while wild-type HgammaS-crystallin is very stable with Tm values of 75.5 and 75.0 degrees C as measured by fluorescence and CD, respectively. Equilibrium unfolding/refolding experiments as a function of GuHCl confirm the relative instability of the G18V mutant. Wild-type HgammaS-crystallin exhibits a two-state transition and reversible refolding above 1.0 M GuHCl, but the unfolding transition of mutant HgammaS-crystallin shows an intermediate state. The first transition (N --> I) shows a [GuHCl](1/2) of 0.5 M while the second transition (I --> U) has the same [GuHCl](1/2) as wild-type HgammaS-crystallin, about 2.0 M. Our present study confirms the high stability of wild-type HgammaS-crystallin and demonstrates that the G18V mutation destabilizes the protein toward heat and GuHCl-induced unfolding. These biophysical characteristics are consistent with the progressive cataract formation seen in the family members carrying this mutation.