Artificial chaperone mediated refolding of xylanase from an alkalophilic thermophilic Bacillus sp. Implications for in vitro protein renaturation via a folding intermediate.
To gain insight into the molecular aspects of unfolding/refolding of enzymes from extremophilic organisms, we have used xylanase from an alkalophilic thermophilic Bacillus as the model system. Kinetics of denaturation/renaturation were monitored using intrinsic fluorescence studies. The protein fluorescence measurements suggested a putative intermediate state present in 0.08 M guanidine hydrochloride with an emission maximum of 345 nm; the far-UV circular dichroism spectra revealed content of secondary structure similar to the native enzyme. Studies with the fluorescent apolar probe 1-anilinonapthalene-8-sulfonate (1,8-ANS) were consistent with the presence of increased hydrophobic surfaces as compared with the native or fully unfolded protein. The refolding of Xyl II, was attempted by a relatively new strategy using an artificial chaperone assisted two-step method. The unfolded xylanase was found to bind to the detergent transiently and the subsequent addition of methyl-beta-cyclodextrin helped to strip the detergent and assist in the folding. Our findings suggested that the detergent stabilized a putative intermediate in the folding pathway seemingly equivalent to the folding state described as molten globule. The reactivation of Xyl II was affected by ionic as well as nonionic detergents. However, the cationic detergent cetyltrimethylammonium bromide (CTAB) provided a maximum reactivation (threefold) of the enzyme. The 'delayed detergent addition' experiments revealed that the detergent acts by suppressing the initial aggregate formation and not by dissolving aggregates. The relevance of our findings to the role of artificial chaperones in vivo is discussed.
Nath
D
D
Biochemical Sciences Division, National Chemical Laboratory, Pashan, Pune, India.
Rao
M
M
D001407:Bacillus / Q000201:enzymology / Q000378:metabolism; D002942:Circular Dichroism; D003505:Cyclodextrins / Q000737:chemistry / Q000378:metabolism; D003902:Detergents / Q000737:chemistry / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018832:Molecular Chaperones / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D020673:Protein Renaturation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry* / Q000378:metabolism*; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D003902:Detergents; D018832:Molecular Chaperones; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D019791:Guanidine