Purpose: The purpose of the present study was to determine the biophysical and chaperone properties of the NH(2)-terminal domain, core domain and COOH-terminal extension of human αA- and αB-crystallins and correlate these properties to those of wild type (WT) αA- and αB-crystallins.
Methods: WT αA- and αB-crystallins cloned into pET 100D TOPO vector, were used as templates to generate different constructs encoding specific regions (NH(2)-terminal domain [NTD], core domain [CD], and COOH-terminal extension, [CTE]). The specific regions amplified by PCR using plasmid DNA from WT αA and WT αB were: αA NTD (residues 1-63), αA CD (residues 64-142), αA CTE (residues 143-173), αB NTD (residues 1-66), αB CD (residues 67-146), and αB CTE (residues 147-175). Resultant blunt-end PCR products were ligated to a pET 100 Directional TOPO vector. DNA sequencing results confirmed the desired constructs. Positive clones were transformed into the BL21 Star (DE3) expression cell line. Protein expression and solubility were confirmed by SDS-PAGE and western blot analysis using a monoclonal antibody against a 6× His-tag epitope. Proteins were purified using Ni(2+)-affinity column chromatography, under native or denaturing conditions, and used for biophysical and chaperone function analyses.
Results: A total of five constructs were successfully generated: αA NTD, αA CD, αB NTD, αB CD, and αB CTE. SDS-PAGE and western blot analyses showed that αA CD and αB CD were present in both the soluble and insoluble fractions, whereas mutant preparations with NTD alone became insoluble and the mutant with CTE alone became soluble. All purified constructs showed alterations in biophysical properties and chaperone function compared to WT α-crystallins. αA NTD and αB CTE exhibited the most notable changes in secondary structural content. Also, αA NTD and all αB-crystallin constructs showed altered surface hydrophobicity compared to their respective WT α-crystallins.
Conclusions: Although the individual α-crystallin regions (i.e., NH(2)-terminal domain, core domain, and COOH-terminal extension) exhibited varied biophysical properties, each region alone retained some level of chaperone function. The NH(2)-terminal domains of αA and αB each showed the maximum chaperone activity of the three regions with respect to their WT crystallins.