In vitro renaturation of alkaline family G/11 xylanase via a folding intermediate: alpha-crystallin facilitates refolding in an ATP-independent manner.
In this study, alkaliphilic family G/11 xylanase from alkali-tolerant filamentous fungi Penicillium citrinum MTCC 6489 was used as a model system to gain insight into the molecular aspects of unfolding/refolding of alkaliphilic glycosyl hydrolase protein family. The intrinsic protein fluorescence suggested a putative intermediate state of protein in presence of 2 M guanidium hydrochloride (GdmCl) with an emission maximum of 353 nm. Here we studied the refolding of GdmCl-denatured alkaline xylanase in the presence and the absence of a multimeric chaperone protein alpha-crystallin to elucidate the molecular mechanism of intramolecular interactions of the alkaliphilic xylanase protein that dictates its extremophilic character. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1- naphthalene sulfonate-binding studies, suggest that alpha-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of xylanase in an ATP-independent manner. A 2 M GdmCl is sufficient to denature alkaline xylanase completely. The hydrodynamic radius (R(H)) of a native alkaline xylanase is 4.0, which becomes 5.0 in the presence of 2 M GdmCl whereas in presence of the higher concentration of GdmCl R(H) value was shifted to 100, indicating the aggregation of denatured xylanase. The alpha-crystallin.xylanase complex exhibited the recovery of functional activity with the extent of approximately 43%. Addition of ATP to the complex did not show any significant effect on activity recovery of the denatured protein.
Dutta
Tanmay
T
Department of Biochemistry, University College of Science, Calcutta University, Kolkata 700 019, West Bengal, India.
Bhattacharjee
Arindam
A
Majumdar
Uddalak
U
Ray
Saugata Sinha
SS
Sahoo
Rupam
R
Ghosh
Sanjay
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000468:Alkalies / Q000378:metabolism*; D043364:Endo-1,4-beta Xylanases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects*; D020673:Protein Renaturation / Q000187:drug effects*; D013050:Spectrometry, Fluorescence; D038201:alpha-Crystallins / Q000494:pharmacology*
D000468:Alkalies; D038201:alpha-Crystallins; D000255:Adenosine Triphosphate; D043364:Endo-1,4-beta Xylanases; D019791:Guanidine