Covalent reinforcement of a fragile region in the dimeric enzyme thymidylate synthase stabilizes the protein against chaotrope-induced unfolding.
Urea and guanidinium chloride induced unfolding of thymidylate synthase, a dimeric enzyme, and engineered interface mutants have been monitored by circular dichroism, fluorescence, and size-exclusion chromatography. Equilibrium unfolding studies show biphasic transitions, with a plateau between 3.5 and 5 M urea, when monitored by far-UV CD and fluorescence energy transfer employing an (aminoethylamino) naphthalenesulfonyl (AEDANS) label at the active site residue, Cys198. AEDANS was also specifically incorporated at position Cys155 in the mutant protein T155C. Direct excitation of this extrinsic fluorophore in the wild type protein (labeled at Cys198) and mutant T155C (labeled at Cys155) showed remarkable differences in the unfolding profiles. C155 AEDANS has a transition centered at 3.5 M urea, which is in contrast to Cys 198 AEDANS (5.5 M urea). Unfolding studies monitored by following intrinsic fluorescence of Trp residues which are located in a small structural domain suggest that this region of the protein is intrinsically fragile. The stable equilibrium intermediate is identified to be an ensemble of partially unfolded aggregated species by gel filtration studies. The chaotrope-induced denaturation of TS appears to proceed through a partially unfolded intermediate that is stabilized by aggregation. Dissociation and loss of structure occur concomitantly at high denaturant concentrations. Introduction of two symmetrically positioned disulfide bridges across the dimer interface in the triple mutant T155C/E188C/C244T (TSMox) stabilized the protein against denaturant-induced unfolding. Aggregate formation was completely abolished in the mutant TSMox, which also enhanced the overall structural stability of the protein. Structural reinforcement of the fragile interface in thymidylate synthase results in dramatic stabilization toward chaotrope-induced unfolding.
Gokhale
R S
RS
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
Agarwalla
S
S
Santi
D V
DV
Balaram
P
P
D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005453:Fluorescence; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008956:Models, Chemical; D008958:Models, Molecular; D015335:Molecular Probes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000378:metabolism*; D014364:Tryptophan; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D015335:Molecular Probes; D011994:Recombinant Proteins; D014364:Tryptophan; D014508:Urea; D013940:Thymidylate Synthase; D019791:Guanidine