title: Refolding of urea-denatured tubulin: recovery of nativelike structure and colchicine binding activity from partly unfolded states.
abstract: Tubulin unfolding in urea proceeds via the formation of a partially unfolded intermediate state, stable in 2 M urea, that unfolds further in higher urea concentrations. The intermediate state had spectroscopic properties reminiscent of a molten globule and negligible colchicine binding activity. Refolding of totally unfolded tubulin in 8 M urea yielded an intermediatelike state characterized by partial burial of tryptophans and partial recovery of secondary and tertiary structures, although colchicine-binding activity of the protein was not regained. Further folding of this intermediatelike state, toward the native conformation, with respect to both structural and functional parameters did not occur. However, a significant percentage of colchicine binding activity and nativelike tertiary structure was recovered when refolding was initiated from partially denatured protein samples, viz., from <1.2 M urea. Thus, although high concentration of urea induced loss of structure and activity was irreversible, the conformational changes induced in restricted regions of tubulin by lower concentrations of urea, which are probably crucial for its various functional properties, could be reversed.
authors.affiliation: Department of Biochemistry, Bose Institute, Centenary Building, Calcutta 700 054, India. bablu@boseinst.ernet.in
authors.forename: S
authors.initials: S
authors.lastname: Guha
authors.forename: B
authors.initials: B
authors.lastname: Bhattacharyya
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000818:Animals; D002942:Circular Dichroism; D003078:Colchicine / Q000378:metabolism*; D019281:Dimerization; D006041:Goats; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism; D014508:Urea / Q000378:metabolism*
chemical_list: D014404:Tubulin; C038518:acrylodan; D014508:Urea; D015081:2-Naphthylamine; D003078:Colchicine