Abstract
Alpha-crystallin, composed of two subunits, alphaA and alphaB, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of alpha-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of alphaA- and alphaB-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of alphaA- and alphaB-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with alphaB showing a higher number of sites than alphaA at 30 degrees C. In agreement with the higher number of hydrophobic sites, alphaB-crystallin demonstrated higher chaperone activity than alphaA at this temperature. Thermodynamic analysis of ANS binding to alphaA- and alphaB-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for alphaA and alphaB at 15 degrees C, alphaA was more potent than alphaB in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of alphaA and alphaB for ANS upon preheating, there was an increase in the number of low affinity sites of alphaA and alphaB. Preheated alphaA, in contrast to alphaB, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of alpha-crystallins, it does not quantitatively correlate with the chaperone function of alpha-crystallins.
Highlights
␣-Crystallin, composed of two subunits, ␣A and ␣B, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions
Thermodynamic analysis of ANS binding to ␣A- and ␣B-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding
There was no change in the number of high affinity binding sites of ␣A and ␣B for ANS upon preheating, there was an increase in the number of low affinity sites of ␣A and ␣B
Summary
Our results indicate that hydrophobicity appears to be a factor in determining the chaperone-like activity of ␣-crystallins, it does not quantitatively correlate with the chaperone function of ␣-crystallins. Unlike ␣B-crystallin, ␣A-crystallin appears to be largely lens-specific Both homopolymers and heteropolymers of ␣-crystallin exhibit chaperone-like activity similar to that of other sHSPs (4 –7). ␣A- and ␣B-crystallins differ in their hydrophobic character with temperature and in their secondary and tertiary structure, molecular size, and other physicochemical properties (14 –16) This makes an exact correlation between hydrophobicity and chaperone activity difficult to establish. Hydrophobicity has been measured qualitatively and quantitatively by spectroscopic methods like fluorescence and equilibrium dialysis, mostly by using two common fluorescent dyes, 8-anilino-1-naphthalenesulfonic acid (ANS) and bis-ANS [19, 20, 22, 24] In such cases, indirect methods such as van’t Hoff analysis for obtaining thermodynamic parameters to elucidate ANS binding are often limited. We correlate hydrophobicity and chaperone activity of ␣A- and ␣B-crystallins at different temperatures to get greater insight into the role of hydrophobicity in the chaperone-like function of ␣-crystallin
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
