Thermodynamic Stability of Human Lens Recombinant αA- and αB-crystallins

Abstract

Lens ␣-crystallin is a 600 -800-kDa heterogeneous oligomer protein consisting of two subunits, ␣A and ␣B. The homogeneous oligomers (␣A-and ␣B-crystallins) have been prepared by recombinant DNA technology and shown to differ in the following biophysical/biochemical properties: hydrophobicity, chaperone-like activity, subunit exchange rate, and thermal stability. In this study, we studied their thermodynamic stability by unfolding in guanidine hydrochloride. The unfolding was probed by three spectroscopic parameters: absorbance at 235 nm, Trp fluorescence intensity at 320 nm, and far-UV circular dichroism at 223 nm. Global analysis indicated that a three-state model better describes the unfolding behavior than a two-state model, an indication that there are stable intermediates for both ␣A-and ␣B-crystallins. In terms of standard free energy (⌬G NU H2O ), ␣A-crystallin is slightly more stable than ␣B-crystallin. The significance of the intermediates may be related to the functioning of ␣-crystallins as chaperonelike molecules.Human lens proteins become progressively less soluble with age and cataract formation. The accumulation of high molecular mass aggregated and insoluble proteins was thought to be the main cause of lens opacity (1, 2). The major crystallin in high molecular mass and insoluble proteins, ␣-crystallin, has been thought to play a major role in the maintenance of lens transparency (3), as manifested by the recent finding that ␣-crystallin acts like a chaperone in in vitro experiments (4). However, the mechanisms of high molecular mass aggregation and insolubilization have not been well established. A reasonable assumption is that these events are related to proteins being in an unstable or not fully folded state, and thus tending to aggregate when their concentrations are high. A study on thermodynamic and kinetic stability may provide a reliable clue.An earlier report indicated that the native ␣-crystallin is thermally very stable (5). It does not denature even at 100°C, but undergoes a thermal transition at 60 -65°C. Later, it was reported that ␣-crystallin becomes partially unfolded at this temperature (6). The refolding is irreversible after exposure to the high temperature (6, 7). On the other hand, the reversibility of denaturant unfolding of ␣-crystallin has been reported (8, 9). The thermodynamic and kinetic stability, however, has not been studied in detail, presumably because of the heterogeneity and oligomeric nature of ␣-crystallin. In contrast, the monomeric ␥-crystallin has been extensively studied (10 -13). Recently, we reported a thermodynamic and kinetic study on ␥F-crystallin (14), which is the most stable ␥-crystallin gene product (15,16).To obtain pure homogeneous ␣-crystallin, we have cloned human lens ␣A-and ␣B-crystallins (17), which are good models for studying the unfolding and refolding properties of ␣-crystallin. In addition to the unfolding intermediate, we also need to consider whether ␣-crystallin has a dissociation intermediate (18). In this work, we used the chemica...

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

“…The standard free energy change of unfolding of ␣A-crystallin at zero urea concentration at 37°C in the absence of ATP is 19.9 kJ/mol. This value of ⌬G 0 compares well with a value of 26.7 kJ/mol reported by Sun et al (39) for the unfolding of ␣A-crystallin at 25°C at infinite dilution of guanidine hydrochloride. In presence of 3 mM ATP at 37°C, ⌬G 0 increases to 24.4 kJ/mol (Table III).…”

Differential Protective Activity of αA- and αB-crystallin in Lens Epithelial Cells

“…Although the activity of ␣B-crystallin is relatively insensitive to temperature, a marked temperature dependence of chaperone activity of ␣A-crystallin has been reported (32)(33)(34)(35)(36). Using several protein substrates, ␣A-and ␣B-crystallin were found to have similar chaperone activities at physiological temperatures (ϳ37°C) when inhibition of lactalbumin aggregation was measured, whereas the chaperone-like activity of ␣B-crystallin was greater than that of ␣A-crystallin when the substrate was alcohol dehydrogenase or insulin (33).…”

“…Recent in vitro studies demonstrate that the chaperone-like activity of ␣-crystallins is sensitive to substrate, buffer conditions, and temperature (32)(33)(34)(35)(36). Although the activity of ␣B-crystallin is relatively insensitive to temperature, a marked temperature dependence of chaperone activity of ␣A-crystallin has been reported (32)(33)(34)(35)(36).…”

“…Using several protein substrates, ␣A-and ␣B-crystallin were found to have similar chaperone activities at physiological temperatures (ϳ37°C) when inhibition of lactalbumin aggregation was measured, whereas the chaperone-like activity of ␣B-crystallin was greater than that of ␣A-crystallin when the substrate was alcohol dehydrogenase or insulin (33). Another factor that may contribute to their different activities is the surface hydrophobicity of the ␣A-and ␣B-crystallins (32)(33)(34)(35)(36). However, neither the differences in vitro chaperone activity toward selected substrate proteins, nor the different surface hydrophobicity of ␣A-and ␣B-crystallin are sufficient to explain the greater anti-apoptotic activity of ␣A-over that of ␣B-crystallin found in the present study (32)(33)(34)(35)(36).…”

An Insight into the Molecular Basis of Salt Tolerance of l-myo-Inositol 1-P Synthase (PcINO1) from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice

“…The C 0.5 of DPcINO1.2 and OsTPcINO1 changed from 3.5 to 3.1 M urea in absence of NaCl to 4.0 and 3.8 M urea in presence of 500 mM NaCl. The standard free energy change (DG 0 ) required to fully unfold these proteins from their native state in absence of urea was computed by fitting the experimental denaturation profile according to a three-state model Sun et al, 1999) as follows: Figure 8. Change in the secondary structure as revealed by CD spectra in OsINO1, PcINO1, and the mutant proteins in the presence of salt.…”