Monitoring of unfolding and refolding in fungal phytase (phyA) by dynamic light scattering.
Role of disulfide bridges in phytase's unfolding-refolding was probed using dynamic light scattering. Phytase was unfolded by guanidinium chloride and then refolded by removing the denaturant by dialysis. Thiol reagents prevented refolding; thus, disulfide bridge formation is an integral step in phytase folding. Catalytic demise of phytase after unfolding and refolding in presence of Tris(2-carboxyethyl)phosphine (TCEP) indicates that disulfide bridges are necessary for refolding. The hydrodynamic radius (rh) of active and unfolded phytase is 4 and 14 nm, respectively. Removal of denaturant through dialysis refolds phytase; its rh shifts back to 4 nm. When TCEP remains in the refolding media, the rh remains high. The unfolded phytase when diluted in assay medium refolds as a function of time at 25 and 37 degrees C, but not at higher temperature. Monitoring rh under denaturing and renaturing condition gives an accurate measure of the folding status of phytase.
Ullah
Abul H J
AH
Southern Regional Research Center, Agricultural Research Services, United States Department of Agriculture, New Orleans, LA 70124, USA. aullah@srrc.ars.usda.gov
Sethumadhavan
Kandan
K
Mullaney
Edward J
EJ
D010832:6-Phytase / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry; D008027:Light; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects; D010720:Phosphines / Q000494:pharmacology; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D012542:Scattering, Radiation; D013696:Temperature
D010832:6-Phytase; D004220:Disulfides; D006859:Hydrogen; D008623:Mercaptoethanol; D010720:Phosphines; C080938:tris(2-carboxyethyl)phosphine