Effect of the Q151X mutation upon the oligomerization of αB-crystallin. The elution profiles of the various αB-crystallins were determined on a 290 × 10-mm Superose 6 column (A). The elution characteristics of the various mutants are plotted relative to those obtained for wild type αB-crystallin (wild type), as highlighted by the vertical line indicating the retention time of the oligomeric complex, which is the major and only peak resolved in the wild type αB-crystallin sample. Additional minor species seen in E164X and 450delA/wild type are highlighted. A summary of the retention times (B) is shown for the major peak resolved during SEC of the wild typeαB-crystallin and other constructs. The retention times of minor peaks are included in the figure as appropriate. The retention times (±S.D.) were calculated from the average of three cycles of SEC. Negative stained images of wild type (C) and Q151XαB-crystallin (D) were obtained by transmission electron microscopy and illustrate the absence of characteristic particles in the Q151X sample, in agreement with the SEC data. Bar, 500 nm.

Thermal stability of wild type and C-terminal extension mutants of αB-crystallin. The effect of the C-terminal extension mutations on the heat-induced aggregation of αB-crystallin was measured at 360 nm over the temperature range 25–86 °C. The temperature was increased at a 1 °C/min over 1 h. Wild type and mutant αB-crystallins were assayed at 0.1 mg/ml. Notice the significantly reduced stability of the E164X, Q151X, and E165X αB-crystallin mutants.

Aggregation of citrate synthase and insulin in the presence of wild type and mutant αB-crystallins. The effect of wild type and mutant αB-crystallin on the thermal aggregation of citrate synthase (A and B), over a 30-min period at 42 °C, and reduction-induced aggregation of insulin (C and D), over a 15-min period at 37 °C was measured using OD₃₆₀ (OD360). A 4:1 molar ratio of substrate:chaperone was used. The relative inhibition of citrate synthase (B) and insulin (D) aggregation by the C-terminal extension mutants was calculated as a percentage of the level of inhibition achieved by wild type αB-crystallin alone (100% inhibition). These data represented the average (±S.D.) of three independent experiments (see supplementary data Figs. S3 and S4 for the complete data sets).

Decreased solubility and increased aggregation potential of Q151X αB-crystallin is revealed by transient transfection into MCF7 cells. A–D, MCF7 cells transiently transfected with wild type (A), Q151X (B), 464delCT (C), or E164X (D) αB-crystallin were fixed at 24 h post-transfection and processed for immunofluorescence microscopy. The subcellular distribution of αB-crystallin and keratin were visualized by double labeling with monoclonal anti-keratin (red channel) and polyclonal anti-αB-crystallin antibodies (green channel). A–D are merged images, showing the superimposition of the green, red, and blue (4′,6-diamidino-2-phenylindole staining) channels. Images are individual optical sections acquired using a confocal laser scanning microscope (Zeiss 510). Cells expressing wild type αB-crystallin (A) showed the expected cytoplasmic distribution of αB-crystallin. In contrast, cells expressing mutant αB-crystallin resulted in the formation of cytoplasmic aggregates of αB-crystallin (B–D). Notice that 464delCT aggregates are scattered throughout the cytoplasm, whereas the Q151X and E164X aggregates locate to the perinuclear region of the cell. Bars, 10 μm. Immunoblotting analysis (E) of αB-crystallin transiently transfected in MCF7 cells shows a shift in solubility for the mutant αB-crystallins. At 24 h post-transfection, cells were extracted with detergent buffer followed by centrifugation at 18,000 × g for 10 min at 4 °C. The resulting supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE followed by immunoblotting analysis using anti-HSP 27 (loading control) and αB-crystallin antibodies. The blot was developed using enhanced chemiluminescence. Whereas wild type αB-crystallin was almost entirely soluble (E, lane 1, labeled S), Q151X (lane 4), 464delCT (lane 6), and E164X (lane 8) were found mostly in the pellet fractions (labeled P) of cell extracts. The relative electrophoretic mobility of mutant αB-crystallins is denoted by the arrowhead (▸). Like wild type αB-crystallin, HSP27 was also largely present in the supernatant fractions (E, lanes 2, 4, and 6, labeled P). These data are representative of three experiments.

Details of the αB-crystallin mutants and their characterization by SDS-PAGE. Structural alignment of the C-terminal extensions of αB-crystallin mutants (A) includes the C-terminal extension from residue 149 (24), and β-sheets and the IX(I/V) motif are indicated as black boxes. The β7 sequence spans residues 118–123 (FHRKYR), β8 sequence spans residues 134–138 (TSSLS), and β9 sequence spans residues 142–148 (VLTVNGP). The highly conserved IX(I/V) motif includes residues 159–161 (IPI). The C-terminal extension consists of residues 149–175 (RKQVSGPERTIPITREEKPAVTAAPKK). B, characterization of the bacterially expressed and purified mutants used in this study by SDS-PAGE. The 450delA (arrow) and 464delCT (arrowhead) mutants could not be produced as individual soluble proteins and therefore were refolded in the presence of wild type αB-crystallin. Molecular weight markers (track M) are indicated (•) in order of increasing relative mobility (40, 33, 24, and 17 kDa). The relative molecular weights of the wild type and mutant αB-crystallins (C) were confirmed by mass spectrometry and compared with values calculated from the amino acid sequences using the Emboss Pepstats program. D, refolding yields for αB-crystallin truncation constructs and mutants purified from inclusion bodies.

Circular dichroism of wild type αB-crystallin and the C-terminal extension mutants. Far UV CD spectra (205–250 nm) for each protein were the average of four spectra. The far UV CD signal was converted to molar ellipticity expressed as degree/cm₂/dmol^–1 to normalize for slight differences in molecular mass between the wild type and the other αB-crystallin constructs. The far UV CD spectra of wild type, A171X, and K174X are similar in shape and magnitude and contained broad minima between 208 and 217 nm. The 450delA/wild type and 464delCT/wild type mixtures show increased negative ellipticity and a shift in minima to 208 nm. Truncation of the C-terminal extension results in the loss of secondary structure reflected by a decrease in negative ellipticity and a shift in wavelength minima (E164X, E165X, Q151X, and Q151X/wild type), with the most dramatic effects seen for Q151X.

Bis-ANS fluorescence binding properties ofαB-crystallin mutants. The effect of the C-terminal mutations upon the relative exposed hydrophobicity was performed using bis-ANS to bind to surface-exposed hydrophobic regions on the mutant and wild type αB-crystallin. Each protein was analyzed at 1 μm in the presence of a 10-fold molar excess of bis-ANS. The excitation wavelength was 410 nm and emission spectra were collected between 420 and 700 nm. All spectra are the average of 10 individual scans. The resulting exposed hydrophobicity is increased for all mutants relative to the wild type αB-crystallin, with the exception of the Q151X mutant.

Sedimentation assay to investigate the prevention of desmin filament aggregation in the presence of various αB-crystallins at 37 °C. A–C shows the % desmin pelleted (A), the % αB-crystallin wild type and mutants remaining soluble in the presence of desmin (B), and the % of the total αB-crystallin pelleted in the presence of desmin (C), following low-speed centrifugation (2500 × g). These data were calculated from SDS-PAGE data of the desmin binding assays. D, an example of the data used to calculate these values for wild type, Q151X/wild type, and Q151X αB-crystallin. These data illustrate the ability of the Q151X αB-crystallin to prevent desmin filament aggregation. Images obtained by electron microscopy of negatively stained samples of the assembled filaments for desmin assembled in the presence of wild type (E) and Q151X (F)αB-crystallin are shown to show that the addition of both chaperones has not dramatically altered filament morphology or length. Notice the lack of crystallin particles in the Q151X sample. Bar, 100 nm. S, supernatant; P, pellet.