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α‐Crystallin binds to the aggregation‐prone molten‐globule state of alkaline protease: Implications for preventing irreversible thermal denaturation
Author(s) -
Tanksale Aparna,
Ghatge Mohini,
Deshpande Vasanti
Publication year - 2002
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.0201802
Subject(s) - molten globule , chemistry , protease , crystallin , chaperone (clinical) , protein folding , denaturation (fissile materials) , biophysics , native state , sodium dodecyl sulfate , biochemistry , crystallography , enzyme , biology , nuclear chemistry , medicine , pathology
Abstract α‐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. α‐Crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The α‐crystallin‐bound I A state exhibited native‐like secondary and tertiary structure showing the interaction of α‐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 α‐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 α‐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 α‐crystallin in the unfolding and refolding of a protease. α‐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.