Agarwalla, Sanjay and Gokhale, Rajesh S and Santi, Daniel V and Balaram, P (1996) Covalent tethering of the dimer interface annuls aggregation in thymidylate synthase. In: Protein Science, 5 (2). pp. 270-277.
Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggregates at concentrations of urea (3.3-5 M), well below that required for complete denaturation, as established by fluorescence and size-exclusion chromatography. In contrast to the wild-type enzyme, an engineered mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are crosslinked by disulfide bridges between residues 155-188‘and 188-155’, does not show this behavior. Aggregation behavior is restored upon disulfide bond reduction in the mutant protein, indicating the involvement of interface segments in forming soluble associated species. Intermolecular disulfide crosslinking has been used as a probe to investigate the formation of larger non-native aggregates. The studies argue for the formation of large multimeric species via a sticky patch of polypeptide from the dimer interface region that becomes exposed on partial unfolding. Covalent reinforcement of relatively fragile protein-protein interfaces may be a useful strategy in minimizing aggregation of non-native structures in multimeric proteins.
|Item Type:||Journal Article|
|Additional Information:||Copyright for this article belongs to Cold Spring Harbor Laboratory Press.|
|Keywords:||intermolecular disulfides;protein aggregation annulment;size-exclusion chromatography;thymidylate synthase|
|Department/Centre:||Division of Biological Sciences > Molecular Biophysics Unit|
|Date Deposited:||04 Nov 2004|
|Last Modified:||19 Sep 2010 04:17|
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