Reversible dissociation and unfolding of the dimeric protein thymidylate synthase.
Conditions for in vitro unfolding and refolding of dimeric thymidylate synthase from Lactobacillus casei were found. Ultraviolet difference and circular dichroism spectra showed that the enzyme was completely unfolded at concentrations of urea over 5.5 M. As measured by restoration of enzyme activity, refolding was accomplished when 0.5 M potassium chloride was included in the refolding mixture. Recombination of subunits from catalytically inactive mutant homodimers to form an active hybrid dimer was achieved under these unfolding-refolding conditions, demonstrating a monomer to dimer association step.
Perry
K M
KM
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Pookanjanatavip
M
M
Zhao
J
J
Santi
D V
DV
Stroud
R M
RM
D002942:Circular Dichroism; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D011189:Potassium Chloride; D014508:Urea; D013940:Thymidylate Synthase