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Hydrophobic core mutations associated with cataract formation destabilize human gammaD‐crystallin
Author(s) -
Drahos Kate Lauren,
King Jonathan
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.850.10
Subject(s) - crystallin , mutant , chemistry , biophysics , amino acid , mutation , protein folding , crystallography , equilibrium unfolding , monomer , biochemistry , biology , gene , organic chemistry , polymer
The human eye lens is composed of fiber cells packed with crystallins up to 400 mg/ml. Human γD‐crystallin (HγD‐Crys) is a monomeric, two‐domain protein found in the lens nucleus. Previous genetic screens for mutations resulting in cataract in mice identified mutations affecting γ‐crystallins. Three such amino acid substitutions were introduced into HγD‐Crys and the mutant proteins L5S, V75D and I90F were expressed and purified from E. coli . All mutant proteins had decreased thermal stability compared with wild type (WT). Equilibrium unfolding/refolding experiments measured the thermodynamic stability of the mutant and WT proteins. WT HγD‐Crys exhibits a three‐state unfolding pathway with the N‐terminal domain (N‐td) unfolding first, followed by the C‐terminal domain (C‐td). L5S and V75D, both N‐td mutants, displayed three‐state unfolding transitions with populated partially‐folded intermediates. In both cases, the first transition was shifted to lower denaturant concentrations. I90F, a C‐td mutant, exhibited a two‐state unfolding transition. In kinetic unfolding experiments the N‐td's of L5S and V75D unfolded faster than WT. I90F was globally destabilized and unfolded through a two‐state mechanism. These results support models of cataract formation in which generation of partially unfolded species are precursors to the aggregated cataractous states responsible for light scattering.