Conformational stability of bovine α-crystallin. Evidence for a destabilizing effect of ascorbate

Abstract

Short-term incubation of bovine alpha-crystallin with ascorbate alters the protein conformational stability. The denaturation curves with urea and guanidinium-chloride show different patterns, suggesting a deviation from a two-state mechanism owing to the presence of one or more intermediates in the unfolding of ascorbate-modified alpha-crystallin. Furthermore, the latter protein profiles are shifted to lower denaturant concentrations indicating a destabilizing action of ascorbate, which is capable of facilitating protein dissociation into subunits as demonstrated by gel filtration with 1.5 M-urea. The decrease in conformational stability cannot be ascribed to any major structural alteration, but rather to localized changes in the protein molecule. In fact, no difference between native and ascorbate-treated alpha-crystallin can be detected by amino acid analysis but perturbation of the tryptophan and tyrosine environment is indicated by alterations in intrinsic fluorescence. Furthermore, turbidity and light-scattering measurements suggest an involvement of the lysine side chains, since aggregability patterns with acetylsalicylic acid are significantly altered. The ascorbate-destabilizing effect on the conformational stability of alpha-crystallin, probably exerted through oxidative modification of amino acid residues and/or the formation of covalent adducts, provokes unfavourable steric interactions between residues along the polypeptide chains, thus favouring aggregation and insolubilization of crystallins which can lead to cataract formation, as also demonstrated by proteolytic digestion patterns which show a lower rate of degradation of the ascorbate-modified alpha-crystallin.

GTP binds to α-crystallin and causes a significant conformational change

“…4. The unfolding profile for ␣-crystallin alone (circles) is consistent with its reported denaturation curve [25]. As shown in Fig.…”

Self-similarity properties of alpha-crystallin supramolecular aggregates

“…Preparation and aggregation of a-crystallin. a-crystallin from bovine eye lens was prepared according to Santini et al (1992). The a-crystallin fractions suspended in 10 mM Tris-HCl buffer, pH 7.4, were thoroughly mixed and pooled together.…”

“…The increase in light scattering in old and cataractous lenses can be ascribed to alterations in protein-water interactions, protein-protein interactions, and lens proteins (Latina et al, 1987;Cooper et al, 1994) due to physicochemical changes of the lens intracellular medium (Delaye et al, 1982;Xia et al, 1994) and/or to age related posttranslational modifications of a-crystallin (Garland et al, 1986;Santini et al, 1992;Luthra and Balasubramanian, 1993;Miesbauer et al, 1994) that disrupt the liquid-like molecular order and promote the formation of large scattering particles (Jedziniak et al, 1978;Guptasarma et al, 1992).…”

Effect of a Lens Protein in Low-Temperature Culture of Novel Immortalized Human Lens Epithelial Cells (iHLEC-NY2)

“…The crystalline lens contains a large amount of CRYAA (HSPB4), which has approximately 55% amino acid homology and similar functions and structures, in addition to αB crystallin. The heat stability and chaperone function of the aggregation of the α-crystallin protein composed of both subunits is deeply involved in maintaining the transparency of the lens [ 37 , 38 , 39 , 40 , 41 , 42 ]. In addition, when the balance of calcium ions (Ca 2+ ) at the molecular level is disrupted, scattered particles with a high molecular weight fraction are formed [ 43 , 44 ], resulting in scattering (opacity) of light in the crystalline lens.…”