Deletion of (54)FLRAPSWF(61) residues decreases the oligomeric size and enhances the chaperone function of alphaB-crystallin

Abstract

AlphaB-crystallin is a member of the small heat shock protein family and is known to have chaperone activity. Using a peptide scan approach, we previously determined that regions 42-57, 60-71, and 88-123 in alphaB-crystallin interact with alphaA-crystallin during heterooligomer formation. To further characterize the significance of the N-terminal domain of alphaB-crystallin, we prepared a deletion mutant that lacks residues (54)FLRAPSWF(61) (alphaBDelta54-61) and found that the absence of residues 54-61 in alphaB-crystallin significantly decreased the homooligomeric mass of alphaB-crystallin. The average oligomeric mass of wild-type alphaB-crystallin and of alphaBDelta54-61, calculated using multiangle light scattering, was 624 and 382 kDa, respectively. The mutant subunits aggregate to form smaller, less-compact oligomers with a 4-fold increase in subunit exchange rate. Deletion of the 54-61 region resulted in a 50% decrease in intrinsic tryptophan fluorescence. The alphaBDelta54-61 mutant showed a 2-fold increase in 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) binding as compared to the wild-type protein, suggesting increased hydrophobicity of the mutant protein. Accompanying the evidence of increased hydrophobicity in the deletion mutant was a 10-fold increase in antiaggregation activity. Homooligomers of 6HalphaA (750 kDa) readily exchanged subunits with alphaBDelta54-61 homooligomers at 37 degrees C, forming heterooligomers with an intermediate mass of 625 kDa. Our data suggest that residues (54)FLRAPSWF(61) contribute to the higher order assembly of alphaB-crystallin oligomers. Residues (54)FLRAPSWF(61) in alphaB-crystallin are not essential for target protein binding during chaperone action, but this region apparently has a role in the chaperone activity of native alphaB-crystallin.

Figure 1

Amino acid sequence of human αB-crystallin showing the deleted ⁵⁴FLRAPSWF⁶¹region: N-terminal domain (green shaded), α-crystallin domain (orange shaded), and C-terminal tail (yellow shaded). The putative αA-crystallin interacting sites and the chaperone site identified by us earlier are also shown.The residues deleted in this study are shown in red.

Figure 2

Dynamic light scattering analysis of wild-type αB-crystallin (solid line, filled circle) and the αBΔ54–61 mutant (broken line, open circle). Proteins (0.15mg in 0.05mL) were incubated at 37 °C for 1 h prior to analysis. Themolarmass distribution across the refractive index peaks, the polydispersity index (PDI), and the hydrodynamic radius of the proteins were analyzed from the MALS data using ASTRA (v5.3.2.10) software. The number of subunits per oligomer was calculated by dividing the oligomeric mass with the mass of the individual subunit.

Figure 3

Transmission electron micrographs of wild-type and mutant αB-crystallins. A drop of 1 mg/mL protein was negatively stained with 2% uranyl acetate and observed under the JEOL 1200EX electron microscope. (A) Wild-type αB-crystallin; (B) αBΔ54–61; (C) αB wild type+ αBΔ54–61 (1:1). Bar on the left corner in panels A, B, and C = 20 nm.

Figure 4

Spectroscopic characterization of wild-type αB-crystallin (solid line) and mutant αBΔ54–61 (broken line). (A) Tryptophan fluorescence intensity. Protein samples of 0.2 mg/mL in phosphate buffer were used. The excitation wavelength was set to 295 nm. (B) Bis-ANS binding. Bis-ANS stock, 10 µL (14.8mM) solution, was added to 0.2 mg/mL protein in phosphate buffer and incubated at 37 °C for 30 min. The samples were excited at 385 nm. (C) Far-UV CD spectra were recorded at a protein concentration of 0.2 mg/mL using a 5 mm path length cell. (D) Near-UV CD spectra were recorded at a protein concentration of 3 mg/mL using a 5 mm path length cell.

Figure 5

Subunit exchange studies of wild-type αB-crystallin and αB-Δ54–61 in the presence and absence of ADH. The labeled proteins were mixed in equal amounts (25 µg) in a total volume of 250 µL of phosphate buffer. To determine the effect of substrate on subunit exchange, ADH (150 µg) was incubated at 37 °C for 15 min prior to the addition of labeled chaperone proteins. The exchange rate is shown beside the graphs.

Figure 6

Demonstration of subunit interaction between 6HαA and αBΔ54–61. (A) Chromatographic profile of 6HαA (250 µg) and Alexa-350-labeled αBΔ54–61 mixture at 0 min time (solid line) and after 3 h (broken line) of incubation. The black lines represent the UV profile at 280 nm. The green lines represent the fluorescence profile. Prior to incubation the bulk of6HαA andαBΔ54–61 elutes at 30 and 35 min, respectively. After 3 h of incubation, the proteins are largely seen coeluting at 33–34 min. (B) SDS–PAGE of fractions collected during chromatography. The tube numbers are offset by minutes due to delay in sample collection.

Figure 7

Comparison of antiaggregation activities of αBΔ54–61 and wild-type αB-crystallins using ADH and CS as substrate proteins. Aggregation of ADH (250 µg) was induced at 37 °C by phosphate buffer containing100mMEDTA, and aggregation of CS (75 µg in40mMHEPES–NaOHbuffer) was initiated by heating the sample at 43 °C. The percentage of substrate protein aggregated in the presence of various concentrations of wild type (solid circle) and αBΔ54–61 (open circle) was calculated at the 45 min time point and plotted. The aggregation of substrate protein in the absence of chaperone protein was considered 100%aggregation. The amount of chaperone protein required to prevent the aggregation of the substrate by 50% was calculated from the nonlinear regression analysis. This value is used to compare the chaperone efficiency of wild-type and mutant proteins.

Figure 8

Molar mass distribution of wild-type αB-ADH(solid line, filled circle) and αB-Δ54–61-ADH (broken line, open circle). Crystallins and the ADH in equal concentration (50 µg) in phosphate buffer were incubated at 37 °Cfor 1 h, and the reaction mixtures were analyzed by MALS following TSK G4000PWxL chromatography. The table below the graph shows additional parameters determined during MALS measurements.