Figure 1.
Crystal structure of βB2-crystallin and sequence alignment of β/γ-crystallins.
(A) Crystal structure of βB2-crystallin (PDB ID: 2BB2). The four subunits are labeled in cyan, green, orange and magenta, respectively. Leu15 at the N-terminus (red) and Trp195 at C-terminus (blue) are highlighted by the space-filling model to show the role of N-terminus in tetramerization of βB2-crystallin. (B) Sequence alignment of the N- and C-termini of β/γ-crystallins. The sequence alignment was performed using the online software MAFFT (http://www.ebi.ac.uk/Tools/msa/mafft/). The sequences used for alignment are: βB1-crystallin (BB1, P53674), βB2-crystallin (BB2, P43320), βB3-crystallin (BB3, P26998), βA3/A1-crystallin (BA3/BA1, P05813), βA2-crystallin (BA2, P53672), βA4-crystallin (BA4, P53673), γA-crystallin (GA, P11844), γB-crystallin (GB, P07316), γC-crystallin (GC, P07315), γD-crystallin (GD, P07320), γN-crystallin (GN, Q8WXF5) and γS-crystallin (GS, P22914).
Figure 2.
Effect of the A2V mutation on βB2-crystallin structure probed by spectroscopic methods.
(A) Far-UV CD spectra. The inset shows SDS-PAGE analysis of the purified recombinant proteins. Lane M is the marker, and the molecular weights of the marker proteins are 170, 130, 95, 72, 55, 43, 34, 26, 17 and 11 kDa, from top to bottom, respectively. The protein concentration for the SDS-PAGE analysis was 1 mg/ml. (B) Near-UV CD spectra. (C) Intrinsic Trp fluorescence with an excitation wavelength of 295 nm. (D) Extrinsic ANS fluorescence with an excitation wavelength of 380 nm. The protein concentration was 0.2 mg/ml. All spectroscopic experiments were performed at 25°C.
Figure 3.
SEC analysis of the WT and mutated βB2-crystallins.
(A) SEC profiles of the WT βB2-crystallin. (B) SEC profiles of the A2V mutant. (C) Protein concentration-dependence of the elution volume of the dimer peak. (D) Protein concentration-dependence of the peak area from the tetramers. In panels A and B, the protein concentration-dependent changes of the peaks are indicated by the arrows. The positions of the standard molecular weight markers are shown at the top of the panels A and B. All samples were equilibrated for 2 h at 4°C before SEC analysis.
Figure 4.
1H-NMR spectra of the WT and mutated βB2-crystallins.
The 500 MHz 1H-NMR spectra were recorded using a protein concentration of 10 mg/ml at 20°C. The chemical shifts were referenced to DSS. The difference spectrum (blue) is obtained by subtracting the NMR spectrum of the mutated protein (red) by that of the WT protein (black). The asterisks indicate the peaks from the buffer.
Figure 5.
Formation of βB2/βA3-crystallin heteromers probed by SEC and native-PAGE analysis.
(A) SEC analysis. Equal molar of βB2- and βA3-crystallin solutions were mixed and injected into the column immediately (0 h) or after 12 h equilibration at 37°C. The peak positions of the dimeric and tetrameric homomers are labeled on the top of the plot. D is dimer, and TT is tetramer. (B) Native-PAGE analysis. βB2- and βA3-crystallin solutions were mixed and equilibrated for 0–16 h at 37°C, and then the mixtures were used for native-PAGE analysis. The red, blue and black arrows indicate the bands corresponding to WT and mutated βB2-, βA3- and βB2/βA3-crystallins, respectively.
Figure 6.
Effects of the A2V mutation on βB2-crystallin structural stability against GdnHCl- or UV-induced denaturation.
(A) Unfolding transition curves from the emission maximum wavelength of the intrinsic Trp fluorescence (Emax). The proteins with a protein concentration of 0.2 mg/ml was denatured in buffer A containing various concentrations of GdnHCl overnight. The raw data were fitted by a two-state transition, and the midpoints of unfolding (Cm) are presented. (B) Concentration-dependence of the UV-irradiation induced aggregation. The samples were irradiated by 254 nm UV light for 24 h at 4°C. The protein concentration (c) for each sample is labeled above the tube, and 0 denotes the buffer in the absence of proteins. (C) Time-course aggregation induced by UV-irradiation. The protein concentration was 1 mg/ml in buffer A.
Figure 7.
Thermal stability of β-crystallins.
(A) Equilibrium thermal transition curves from Emax. The data were fitted by a two-state model, and the midpoints of unfolding (Tm) are shown in the plot. The protein solutions were heated continuously by a water bath from 28°C to 86°C, and fluorescence spectra were recorded every 2°C after 2 min equilibration at the given temperature. (B) Concentration-dependence of the thermal aggregation kinetics. Only the representative kinetic data are presented. The protein solutions were heated at 70°C continuously, and the turbidity data were recorded every 2 s. (C) Relationship between the maximum turbidity and protein concentration. The data were fitted by two linear parts, and the fitting results are shown by lines. The turbidity values above 1.5 were not included in the fitting due to the limitations of the technique. (D) Protection of βA3-crystallin thermal aggregation by βB2-crystallin at 50°C (solid lines) or 55°C (dotted lines). The βB2/βA3-crystallin heteromer was prepared by incubating the mixtures containing equal molar of βB2- and βA3-crystallins for 20 h at 37°C.
Figure 8.
Aggregation of β-crystallins during kinetic refolding.
(A) Time-course study of the aggregation of β-crystallins with a final concentration of 0.2 mg/ml. The proteins were denatured by 4 M GdnHCl for 12 h, and refolding is initiated by fast manual dilution (1∶40) of the denatured proteins in buffer A. The dead time of the aggregation experiments was 2 s. (B) Characterization of the morphology of the aggregates formed after 10 min refolding by EM. The bars in the pictures represent 100 nm. The positions of typical aggregates are labeled by open squares.