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Covalent tethering of the dimer interface annuls aggregation in thymidylate synthase
S Agarwalla1, R S Gokhale, D V Santi
1Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
Protein Science : a Publication of the Protein Society
|February 1, 1996
Summary
Thymidylate synthase (TS) aggregation is prevented in a disulfide-crosslinked mutant, revealing insights into protein assembly. This suggests stabilizing protein interfaces can inhibit harmful aggregation.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Thymidylate synthase (TS) is a crucial dimeric enzyme in DNA synthesis.
- Proteins can form soluble aggregates during partial unfolding, impacting their function.
- Understanding protein aggregation mechanisms is vital for preventing disease and improving protein stability.
Purpose of the Study:
- To investigate the aggregation behavior of thymidylate synthase (TS) under denaturing conditions.
- To explore the role of protein-protein interfaces in the formation of soluble aggregates.
- To assess the potential of disulfide crosslinking in preventing non-native protein aggregation.
Main Methods:
- Utilized fluorescence spectroscopy and size-exclusion chromatography to monitor protein aggregation.
- Engineered a mutant thymidylate synthase (TSMox) with intermolecular disulfide crosslinks.
- Investigated aggregation by reducing disulfide bonds in the TSMox mutant.
Main Results:
- Wild-type thymidylate synthase (TS) forms soluble aggregates in 3.3-5M urea.
- The disulfide-crosslinked TSMox mutant does not exhibit this aggregation behavior.
- Aggregation was restored in the TSMox mutant upon reduction of disulfide bonds, highlighting the role of interface segments.
Conclusions:
- Partial unfolding exposes interface regions, leading to the formation of large multimeric species.
- Intermolecular disulfide crosslinking effectively prevents aggregation of thymidylate synthase.
- Covalent stabilization of protein interfaces may be a viable strategy to inhibit aggregation in multimeric proteins.