Alpha-crystallin binds to the aggregation-prone molten-globule state of alkaline protease: implications for preventing irreversible thermal denaturation

Abstract

Alpha-crystallin, the major eye-lens protein with sequence homology with heat-shock proteins (HSPs), acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain more insight into its chaperoning ability, we used a protease as the model system that is known to require a propeptide (intramolecular chaperone) for its proper folding. The protease ("N" state) from Conidiobolus macrosporus (NCIM 1298) unfolds at pH 2.0 ("U" state) through a partially unfolded "I" state at pH 3.5 that undergoes transition to a molten globule-(MG) like "I(A)" state in the presence of 0.5 M sodium sulfate. The thermally-stressed I(A) state showed complete loss of structure and was prone to aggregation. Alpha-crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The alpha-crystallin-bound I(A) state exhibited native-like secondary and tertiary structure showing the interaction of alpha-crystallin with the MG state of the protease. 8-Anilinonaphthalene sulphonate (ANS) binding studies revealed the involvement of hydrophobic interactions in the formation of the complex of alpha-crystallin and protease. Refolding of acid-denatured protease by dilution to pH 7.5 resulted in aggregation of the protein. Unfolding of the protease in the presence of alpha-crystallin and its subsequent refolding resulted in the generation of a near-native intermediate with partial secondary and tertiary structure. Our studies represent the first report of involvement of a molecular chaperone-like alpha-crystallin in the unfolding and refolding of a protease. Alpha-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of the alkaline protease and keeps it in a near-native, folding-competent intermediate state.

Fig. 1.

Circular dichroism (CD) spectra of the alkaline protease from Conidiobolus (APC): (A) Near-UV and (B) far-UV CD spectra of the enzyme at concentrations 5.5 and 0.6 μM, respectively. (1) "N" state, (2) "I" state, and (3) "U" state.

Fig. 2.

pH dependence of the fluorescence intensity of APC: (A) Fluorescence intensity of APC (0.3 μM) was monitored at 340 nm in the absence (○) and presence (•) of 0.5 M Na₂SO₄ at 25°C. (B) Fluorescence emission spectra of APC (excitation 295 nm). (——) "N" state, (••-••-••) "I" state, (. . .) "U" state, and (- - - -) "I_A" state.

Fig. 3.

Exposure of hydrophobic surfaces of APC on acid-induced unfolding measured by 8-anilinonaphthalene sulphonate (ANS) binding: (A) ANS (20 μM) was added to the enzyme (0.3μM) and incubated for 20 min at 25°C in the absence (○) and presence (•) of 0.5 M Na₂SO₄. (B) ANS fluorescence emission spectra of APC (excitation at 369 nm). (1) "N" state, (2) "N" state in the presence of 0.5 M Na₂SO₄, (3) "I" state, (4) "U" state, (5) "I_A" state, and (6) U state in the presence of 0.5 M Na₂SO₄.

Fig. 4.

Thermal cooperativity of APC at different pH values: The effect of increasing temperatures on shifts in the emission maximum of APC was assessed by monitoring the I _330/350 ratio at pH 7.5 (), pH 5.0 (▪), pH 3.5 (), pH 2.0 (▴), and pH 3.5 in the presence of 0.5 M Na₂SO₄ (○).

Fig. 5.

Interaction of the chaperone α-crystallin with APC: (A) The effect of α-crystallin on thermally induced aggregation of the I_A state. The I_A state of APC (0.3 μM) (1) at 58°C and (2) in the presence of α-crystallin. (B) Far-UV CD spectra of the (1) N state, (2) I_A state, (3) I_A state at 58°C, and (4) α-crystallin-bound I_A state. (C) Trp fluorescence of (1) N state, (2) U state, (3) I_A state at 58°C, and (4) α-crystallin-bound I_A state.

Fig. 6.

Interaction of the chaperone α-crystallin with APC: (A) Fluorescence spectra of the isoatoic anhydride-labeled APC (1) native enzyme, (2) α-crystallin bound I_A state, and (3) I_A state at 58°C. (B) ANS fluorescence spectra of (1) α-crystallin at 58°C, (2) I_A state of APC added to α-crystallin at 58°C, (3) I_A state of APC at 58°C, (4) complex of α-crystallin and I_A state cooled to 42°C, (5) cooled to 26°C, and (6) mixture of α-crystallin and I_A state at 26°C.

Fig. 7.

Refolding of APC in the presence of α-crystallin: (A) Trp fluorescence of APC (1) N state, (2) APC unfolded in the presence of α-crystallin and refolded by dilution in the presence of 0.5 M Na₂SO₄ at pH 7.5, and (3) U state. (B) ANS fluorescence spectra of APC unfolded and refolded in the (1) absence and (2) presence of α-crystallin. (C) Far-UV CD spectra of (1) native APC and (2) APC unfolded in the presence of α-crystallin and refolded by dilution to pH 7.5 in the presence of salt.