The G18V CRYGS mutation associated with human cataracts increases gammaS-crystallin sensitivity to thermal and chemical stress

Abstract

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.

Figure 1

sedimentation equilibrium data obtained for wild type and mutant HγS-crystallins at 20°C. Absorbance (B, D) and residuals (A,C) ploted for wild type and mutant HγS-crystallins. Opened circles showed the protein concentration profile represented by the UV absorbance gradients in the centrifuge cell at 280 nm. The solid lines indicated the calculated fit for monomer.

Figure 2

Far-UV CD spectra of wild type (top) and G19V mutant (bottom) γS-crystallin in increasing concentrations of GuHCl.

Figure 3

Fluorescence emission spectra of wild type (top) and G18V mutant (bottom) γS-crystallin in increasing concentrations of GuHCl.

Figure 4

ANS emission spectra of wild type and G18V HγS-crystallin in the presence of 0, 1, and 2M GuHCI.

Figure 5

Thermal denaturation curves of wild type and G18V mutant HγS-crystallins. A. Tryptophan fluorescence B. CD at 218 nm.

Figure 6

Plots of the ΔG values of wild type and G18V mutant γS-crystallin as a function of temperature and Calculation the the van’t Hoff equation: d(lnK)/d(1/T) = -ΔH/R. A. ΔG plot from tryptophan fluorescence in figure 5A B. van’t Hoff plot from tryptophan fluorescence in figure 5A C. ΔG plot from CD at 218 nm in figure 5B D. van’t Hoff plot from CD 218 nm in figure 5B.

Figure 7

GuHCl-induced equilibrium unfolding/refolding of wild type and mutant HγS-crystallin