Covalent Reinforcement of a Fragile Region in the Dimeric Enzyme Thymidylate Synthase Stabilizes the Protein against Chaotrope-Induced Unfolding

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• 作者：Rajesh S. Gokhale ; Sanjay Agarwalla ; Daniel V. Santi ; and Padmanabhan Balaram
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：June 4, 1996
• 年：1996
• 卷：35
• 期：22
• 页码：7150 - 7158
• 全文大小：430K
• 年卷期：v.35,no.22(June 4, 1996)
• ISSN：1520-4995

文摘

Urea and guanidinium chloride induced unfolding of thymidylatesynthase, a dimeric enzyme,and engineered interface mutants have been monitored by circulardichroism, fluorescence, and size-exclusion chromatography. Equilibrium unfolding studies showbiphasic transitions, with a plateau between3.5 and 5 M urea, when monitored by far-UV CD and fluorescence energytransfer employing an(aminoethylamino)naphthalenesulfonyl (AEDANS) label at the activesite residue, Cys198. AEDANSwas also specifically incorporated at position Cys155 in the mutantprotein T155C. Direct excitation ofthis extrinsic fluorophore in the wild type protein (labeled at Cys198)and mutant T155C (labeled atCys155) showed remarkable differences in the unfolding profiles.C155 AEDANS has a transition centeredat 3.5 M urea, which is in contrast to Cys 198 AEDANS (5.5 M urea).Unfolding studies monitored byfollowing intrinsic fluorescence of Trp residues which are located in asmall structural domain suggestthat this region of the protein is intrinsically fragile. Thestable equilibrium intermediate is identified tobe an ensemble of partially unfolded aggregated species by gelfiltration studies. The chaotrope-induceddenaturation of TS appears to proceed through a partially unfoldedintermediate that is stabilized byaggregation. Dissociation and loss of structure occurconcomitantly at high denaturant concentrations.Introduction of two symmetrically positioned disulfide bridgesacross the dimer interface in the triplemutant T155C/E188C/C244T (TSMox) stabilized the protein againstdenaturant-induced unfolding.Aggregate formation was completely abolished in the mutant TSMox,which also enhanced the overallstructural stability of the protein. Structural reinforcement ofthe fragile interface in thymidylate synthaseresults in dramatic stabilization toward chaotrope-inducedunfolding.