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Mistranslation fragment of an in silico designed novel-fold protein forms and exceptionally stable symmetric homodimer with a high-affinity interface


EMSL Project ID
16722

Abstract

We recently used computational protein design to create an extremely stable, globular protein, Top7, with a sequence and fold not observed previously in nature. Since Top7 was created in the absence of genetic selection, it provides a rare opportunity to investigate aspects of the cellular protein production and surveillance machinery that are subject to natural selection. Here we show that a portion of the Top7 protein corresponding to the final 49 C-terminal residues is efficiently mistranslated and accumulates at high levels in E. coli. We used circular dichroism spectroscopy, size-exclusion chromatography, small-angle x-ray scattering, analytical ultra-centrifugation, and NMR spectroscopy to show that the resulting CFr protein adopts a compact, extremely-stable, obligate, symmetric, homo-dimeric structure. Based on the solution structure, we engineered an even more stable variant of CFr by disulfide-induced covalent circularisation that should be an excellent platform for design of novel functions. The accumulation of high levels of CFr exposes the high error rate of the protein translation machinery, and the rarity of correspondingly stable fragments in natural proteins implies a stringent evolutionary pressure against protein sub-fragments that can independently fold into stable structures. The symmetric self-association between two identical mistranslated CFr sub-units to generate an extremely stable structure parallels a mechanism for natural protein-fold evolution by modular recombination of stable protein sub-structures.

Project Details

Project type
Capability Research
Start Date
2005-10-25
End Date
2006-02-22
Status
Closed

Team

Principal Investigator

Gabriele Varani
Institution
University of Washington

Team Members

David Baker
Institution
University of Washington

Related Publications

Dantas G, AL Watters, B Lunde, Z Eletr, NG Isern, T Roseman, J Lipfert, S Doniach, M Tompa, B Kuhlman, BL Stoddard, G Varani, and D Baker. 2006. "Mis-translation of a Computationally Designed Protein Yields an Exceptionally Stable Homodimer: Implications for Protein Engineering and Evolution.." Journal of Molecular Biology 362(5):1004-1024.
Dobson N, G Dantas, D Baker, and G Varani. 2006. "High-Resolution Structural Validation of the Computational Redesign of Human U1A Protein ." Structure with Folding & Design 14(5):847-856.