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-Crystallin binds to the aggregation-prone molten-globule state of alkaline protease: Implications for preventing irreversible thermal denaturation

¹ Levine Science Research Center, Duke University, Durham, North Carolina 27708, USA
² Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India

Reprint requests to: Vasanti Deshpande, Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India; e-mail: vasanti{at}dalton.ncl.res.in, vasantil2000{at}yahoo.com; fax: 91-20-5884032.

-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. -Crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The -crystallin-bound I_A state exhibited native-like secondary and tertiary structure showing the interaction of -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 -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 -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 -crystallin in the unfolding and refolding of a protease. -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.

Keywords: Protease; molten globule; -crystallin; thermal denaturation; aggregation; protein folding

Abbreviations: APC, alkaline protease from Conidiobolus • ANS, 8-anilinonaphthalene sulphonate • SAAPF-pNA, N-succinyl-ala-ala-pro-phenylala-p-nitroanilide

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