Abstract
Purpose: :
An autosomal dominant cortical progressive cataract is associated with a G18V mutation in γS-crystallin. Here we investigate changes in the conformation and stability of human γS-crystallin (HγS) resulting from the G18V mutation.
Methods: :
Recombinant WT and G18V mutant HγS was cloned into pET-20b(+) and expressed in E. Coli and purified by ion-exchange and size-exclusion chromatography. Far- and near-UV CD and intrinsic tryptophan fluorescence spectra of wild type and G18V HγS were obtained in increasing concentrations of guanidinium hydrochloride (GuHCI). Thermostability was monitored by recording fluorescence emission at 328nm and 355nm at a scan rate of 1.0°C/min. Intrinsic tryptophan fluorescence was used to characterize the unfolding-refolding equilibrium of wild type and G18V HγS as a function of GuHCI concentration at neutral pH and 25°C.
Results: :
No gross changes in the secondary structure of the G18V mutant are observed under normal conditions as judged by far-UV CD. In the presence of increasing GuHCl, a sharp red shift in the fluorescence max and an increase in emission as exposure of tryptophan to the surface increases occur earlier in the G18V mutant. When subjected to thermal denaturation, the G18V HγS is obviously less stable than wild type. The mutant begins to denature above 42°C in a biphasic manner while wild type is very stable with a single phase Tm value of 75.5°C. Equilibrium unfolding/refolding experiments also confirms the instability of G18V HγS. Wild type HγS exhibits a two-state transition and reversible refolding above 1.0M GuHCI, but a plateau is revealed in the unfolding transition of the G18V HγS, which undergoes the first transition (N→I) with a [GdmCl]1/2 of 0.5 M and the second transition (I→U) the same as wild type with a [GdmCl]1/2 of about 2.0 M.
Conclusions: :
Our present study, further confirms the high stability of wild type HγS, and shows that the G18V mutation leads to destabilization of the protein towards heat and GuHCI -incduced unfolding. These are consistent with and explain the progressive cataract formation noted in affected individuals in this family.
Keywords: cataract • crystallins • protein structure/function