Folding and stability of the isolated Greek key domains of the long-lived human lens proteins gammaD-crystallin and gammaS-crystallin

Abstract

The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric gamma-crystallins and oligomeric beta-crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human gammaD-crystallin revealed that the C-terminal domain folds first and nucleates the folding of the N-terminal domain. This result suggested that the human N-terminal domain might not be able to fold on its own. We constructed and expressed polypeptide chains corresponding to the isolated N- and C-terminal domains of human gammaD-crystallin, as well as the isolated domains of human gammaS-crystallin. Both circular dichroism and fluorescence spectroscopy indicated that the isolated domains purified from Escherichia coli were folded into native-like monomers. After denaturation, the isolated domains refolded efficiently at pH 7 and 37 degrees C into native-like structures. The in vitro refolding of all four domains revealed two kinetic phases, identifying partially folded intermediates for the Greek key motifs. When subjected to thermal denaturation, the isolated N-terminal domains were less stable than the full-length proteins and less stable than the C-terminal domains, and this was confirmed in equilibrium unfolding/refolding experiments. The decrease in stability of the N-terminal domain of human gammaD-crystallin with respect to the complete protein indicated that the interdomain interface contributes of 4.2 kcal/mol to the overall stability of this very long-lived protein.

Figure 1.

Crystal structure of γD_WT and NMR structure of murine γS_WT. Both full-length proteins are ∼20 kDa in size. (A) A ribbon diagram of the γD_WT X-ray crystal structure (PDB ID: 1HK0) (Basak et al. 2003). The isolated γD_N protein ends at Pro82 (highlighted in dark gray) not including the short interdomain linker. The isolated γD_C protein begins at Arg 89 (highlighted in dark gray) at the beginning of the β-sheet. (B) A ribbon diagram representing the NMR structure of the murine γS-crystallin protein (PDB ID: 1ZWO) (Wu et al. 2005). The His 86 position is the same amino acid in the human sequence, while the Ala 93 position is replaced with Tyr in the human sequence. Sequence alignment between human and murine γS-crystallin shows 89% identity and 96% similarity (bl2seq, Blosum 62 matrix).

Figure 2.

Amino acid sequence alignment of γD_WT and γS_WT. The regions of the proteins included in the isolated domain proteins are highlighted, (blue) γD_N G1–P82, (red) γD_C R89–S174, (green) γS_N S1–H86, (gray) γS_C Y93–E177. Upper numbers represent the residues in γD_WT, and lower numbers represent the residues in γS_WT.

Figure 3.

Analytical size exclusion chromatography profiles of the isolated domains and wild-type proteins. All samples were loaded onto a Superdex 200 10/300 GL column at a protein concentration of 80 μg/mL. The molecular weight standards were ovalbumin (45 kDa), chymotrypsinogen A (25 kDa), ribonuclease A (13.7 kDa), elution volumes of which are depicted by arrows. (A) γD_WT, γD_N, and γD_C; (B) γS_WT, γS_N, and γS_C. The secondary peak seen in the γD_N sample was variable and usually less prominent and has not been identified.

Figure 4.

Far-UV CD spectra of isolated domains and full-length proteins. Samples are at a protein concentration of 100 μg/mL in 10 mM sodium phosphate buffer, pH 7.0 at 37°C. (A) CD spectra recorded from 195–260-nm wavelengths for (black ♦) γD_WT, (blue •) γD_N, and (red ▪) γD_C. (B) CD spectra recorded from 195–260-nm wavelengths for (blue ▴) γS_WT, (green ) γS_N, and (orange ) γS_C.

Figure 5.

Fluorescence emission spectra of native and unfolded isolated domains and full-length γD_WT and γS_WT. All proteins were excited at 295 nm and emissions were recorded from 310 to 400 nm. Samples consisted of 10 μg/mL protein in 100 mM sodium phosphate, 1 mM EDTA, 5 mM DTT, pH 7.0, and 5.5 M GuHCl for unfolded samples equilibrated at 37°C. (A) Native spectra for γD_WT (♦) and unfolded spectra for γD_WT (line). (B) γD_N native (•) and γD_N unfolded (line). (C) γD_C native (▪) and γD_C unfolded (line). (D) γS_WT native (▴) and γS_WT unfolded (line). (E) γS_N native (◢) and γS_N unfolded (line). (F) γS_C native (◣) and γS_C unfolded (line).

Figure 6.

Thermal denaturation of γD_WT, γS_WT, and their individual domains. Samples were prepared at 100 μg/mL protein concentration in 10 mM sodium phosphate buffer (pH 7.0). CD signal at 218 nm was monitored as the temperature was increased from 25°C to 90°C. Data were normalized and the native fraction was calculated (see Materials and Methods). (A) (♦) γD_WT, (•) γD_N, and (▪) γD_C. (B) (▴) γS_WT, (◢)γS_N, and (◣) γS_C.

Figure 7.

Equilibrium unfolding (closed symbols) and refolding (open symbols) transitions of (♦) γD_WT, (•) γD_N, and (▪) γD_C. Samples contained 10 μg/mL protein, 100 mM sodium phosphate, 1 mM EDTA, 5 mM DTT (pH 7.0), and various concentrations of GuHCl at 37°C. The ratios of fluorescence emission at 360 nm and 320 nm were calculated. Equilibrium data fits are indicated by solid black lines.

Figure 8.

Equilibrium unfolding (closed symbols) and refolding (open symbols) transitions for (▴) γS_WT, (◢) γS_N, and (◣) γS_C. Samples contained 10 μg/mL protein, 100 mM sodium phosphate, 1 mM EDTA, 5 mM DTT (pH 7.0), and various concentrations of GuHCl at 37°C. The ratios of fluorescence emission at 360 nm and 320 nm were calculated and plotted versus the concentration of GuHCl. Two-state fits of the equilibrium data are indicated by solid black lines.

Figure 9.

Kinetic refolding of γD_WT (inset shows completion of γD_WT refolding kinetics reaction), γD_N, and γD_C. The proteins were unfolded at high GuHCl concentration, then diluted into 100 mM sodium phosphate, 1 mM EDTA, 5 mM DTT (pH 7.0) buffer for a final protein concentration of 10 μg/mL. Protein tryptophan fluorescence emission at 350 nm was recorded every second, and data were normalized for comparison. All experiments were performed at 18°C. The final GuHCl concentrations were 1 M for γD_WT and 0.55 M for γD_N, and γD_C (see text for details).

Figure 10.

Kinetic refolding of isolated domains of γS_WT (inset shows completion of γS_WT refolding kinetics reaction), γS_N, and γS_C. Protein was unfolded at high GuHCl concentration, then diluted into 100 mM sodium phosphate, 1 mM EDTA, 5 mM DTT (pH 7.0) buffer for a final protein concentration of 10 μg/mL. Protein tryptophan fluorescence emission at 350 nm was recorded every second, and data were normalized for comparison. All experiments were performed at 18°C. The final GuHCl concentration was 0.55 M for γS_WT, γS_N, and γS_C (see text for details).