title: 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.
authors.affiliation: Levine Science Research Center, Duke University, Durham, NC 27708, USA.
authors.forename: Aparna
authors.initials: A
authors.lastname: Tanksale
authors.forename: Mohini
authors.initials: M
authors.lastname: Ghatge
authors.forename: Vasanti
authors.initials: V
authors.lastname: Deshpande
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002942:Circular Dichroism; D020092:Conidiobolus / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism*; D004795:Enzyme Stability / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D018832:Molecular Chaperones / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation / Q000502:physiology; D017510:Protein Folding; D020673:Protein Renaturation; D038201:alpha-Crystallins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000817:Anilino Naphthalenesulfonates; D018832:Molecular Chaperones; D038201:alpha-Crystallins; C027132:1-anilino-8-naphthalenesulfonate; D010450:Endopeptidases