期刊
PROTEIN SCIENCE
卷 26, 期 6, 页码 1138-1149出版社
WILEY
DOI: 10.1002/pro.3164
关键词
intermediate state; folding; misfolding; aggregation; amyloid fibrils; worm-like fibrils; disulfide bonds
资金
- Instituto de Salud Carlos III/FEDER [FIS-PI-13-01330]
- Generalitat de Catalunya [SGR-GRC-2014-00885]
- Generalitat de Catalunya/FEDER [2014-PROD00032]
- PIF-UAB Student Grant
ScFv-h3D6 is a single chain variable fragment that precludes A beta peptide-induced cytotoxicity by withdrawing A beta oligomers from the amyloid pathway to the worm-like pathway. Production of scFv molecules is not a straightforward procedure because of the occurrence of disulfide scrambled conformations generated in the refolding process. Here, we separately removed the disulfide bond of each domain and solved the scrambling problem; and then, we intended to compensate the loss of thermodynamic stability by adding three C-terminal elongation mutations, previously described to stabilize the native fold of scFv-h3D6. Such stabilization occurred through stabilization of the intermediate state in the folding pathway and destabilization of a different, beta-rich, intermediate state driving to worm-like fibrils. Elimination of the disulfide bridge of the less stable domain, V-L, deeply compromised the yield and increased the aggregation tendency, but elimination of the disulfide bridge of the more stable domain, V-H, solved the scrambling problem and doubled the production yield. Notably, it also changed the aggregation pathway from the protective worm-like morphology to an amyloid one. This was so because a partially unfolded intermediate driving to amyloid aggregation was present, instead of the beta-rich intermediate driving to worm-like fibrils. When combining with the elongation mutants, stabilization of the partially unfolded intermediate driving to amyloid fibrils was the only effect observed. Therefore, the same mutations drove to completely different scenarios depending on the presence of disulfide bridges and this illustrates the relevance of such linkages in the stability of different intermediate states for folding and misfolding.
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