title: Unfolding and Refolding Study of a Large Dimeric Protein β-Glucosidase from Almond Monitored by Fluorescence Spectroscopy.
abstract: In our present investigation, the unfolding and refolding of β-glucosidase (BGL-Al) from sweet almond was investigated using tryptophan (Trp) fluorescence spectroscopy. When the unfolding of BGL-Al was induced by guanidium chloride (GdnHCl) and monitored using biological activity as well as Trp fluorescence spectroscopic measurement, we observed that the denaturation of BGL-Al could be easily induced by low concentration of GdnHCl and the enzyme was completely inactivated at 1.0 M GdnHCl. Higher unfolding in the presence of reducing agent revealed that the protein perhaps containing multiple di-sulfide bonds indicating a reason of high stability against unfolding by GdnHCl. Refolding results suggested that the protein refolded with high yield from 1 M GdnHCl denatured state, however, refolded with negligible yield from completely unfolded state. The kinetic studies of BGL-Al refolding unravel a two phase refolding process with calculated t1/2 (refolding half time) of 1.8 and 33 min, respectively. When 8-Anilino-1-naphthalenesulfonic acid (ANS) was used as extrinsic fluorophore, we found that the surface hydrophobicity of BGL-Al was continuously decreased during GdnHCl-mediated unfolding. The surface hydrophobicity of the protein was calculated to be as high as 128.32. Acrylamide quenching study demonstrated that Trp residues of BGL-Al are mostly and hence they must be located either on the surface or in the crevices accessible by quenchers.
authors.forename: Kanti K
authors.initials: KK
authors.lastname: Yadav
authors.affiliation: Structural Biology and Nanomedicine Laboratory, Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela-769008, Odisha, India. spaul@nitrkl.ac.in.
authors.forename: Subhankar
authors.initials: S
authors.lastname: Paul
mesh_terms: D019791:Guanidine / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D055503:Protein Multimerization / Q000187:drug effects; D058849:Protein Refolding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D058767:Protein Unfolding / Q000187:drug effects; D000068181:Prunus dulcis / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D001617:beta-Glucosidase / Q000737:chemistry*
chemical_list: D001617:beta-Glucosidase; D019791:Guanidine