Refolding of glutamate dehydrogenase from Bacillus acidocaldarius after guanidinium chloride-induced unfolding.
The hexameric NAD-dependent glutamate dehydrogenase from the thermophilic eubacterium Bacillus acidocaldarius is the first glutamate dehydrogenase which spontaneously refolds in vitro. After 24 h unfolding in 6 M guanidinium chloride at 20 degrees C, refolding was achieved by dilution of the denaturant. The yield of reconstitution obtained in the presence of 1,4 dithio-DL-threitol in the unfolding/refolding mixture was 75%. The unfolding/refolding equilibria have been studied by fluorescence, circular dichroism and catalytic activity, which was 50% inhibited at 0.08 M guanidinium chloride, a value 30-fold lower than the transition midpoint detected by physical changes. Refolding was attempted in the presence of various additives, at different temperatures and varying enzyme and residual guanidinium chloride concentration. The refolded enzyme, after removal of inactive aggregated species, completely resembles the native enzyme in term of its physicochemical and kinetic properties as well as thermophilicity.
Consalvi
V
V
Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università La Sapienza, Roma, Italy.
Millevoi
S
S
Chiaraluce
R
R
de Rosa
M
M
Scandurra
R
R
D001407:Bacillus / Q000201:enzymology*; D002942:Circular Dichroism; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D006146:Guanidines; D046911:Macromolecular Substances; D009243:NAD; D005969:Glutamate Dehydrogenase; D019791:Guanidine