Mis-translation of a Computationally Designed Protein Yields an Exceptionally Stable Homodimer: Implications for Protein Engineering and Evolution

Mis-translation of a Computationally Designed Protein Yields an Exceptionally Stable Homodimer: Implications for Protein Engineering and Evolution
Received 17 May 2006; revised 21 July 2006; accepted 29 July 2006. Edited by F. Schmid. Available online 4 August 2006.
Gautam Dantas1, 1, Alexander L. Watters2, Bradley M. Lunde3, Ziad M. Eletr7, Nancy G. Isern8, Toby Roseman4, Jan Lipfert9, Sebastian Doniach9, Martin Tompa4, Brian Kuhlman7, Barry L. Stoddard10, Gabriele Varani1, 5 and David Baker1, 6
Journal of Molecular Biology
Volume 362, Issue 5 , 6 October 2006
Copyright ? 2006 Elsevier Ltd All rights reserved.
1Department of Biochemistry, University of Washington, Seattle 98195, USA
2Department of Molecular and Cellular Biology, University of Washington, Seattle 98195, USA
3Bio-Molecular Structure and Design Program, University of Washington, Seattle 98195, USA
4Department of Computer Science and Engineering, University of Washington, Seattle 98195, USA
5Department of Chemistry, University of Washington, Seattle 98195, USA
6Howard Hughes Medical Institute, University of Washington, Seattle 98195, USA
7Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA
8EMSL High Field Molecular Resonance Facility, PNNL, Richland, WA 99352, USA
9Department of Physics, Stanford University, Stanford CA 94305, USA
10Division of Basic Sciences, Fred Hutchison Cancer Research Center, 1100 Fairview Ave N, Seattle, WA 98109, USA
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 mis-translated and accumulates at high levels in Escherichia coli. We used circular dichroism, size-exclusion chromatography, small-angle X-ray scattering, analytical ultra-centrifugation, and NMR spectroscopy to show that the resulting C-terminal fragment (CFr) protein adopts a compact, extremely stable, 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. The rarity of correspondingly stable fragments in natural proteins coupled with the observation that high quality ribosome binding sites are found to occur within E. coli protein-coding regions significantly less often than expected by random chance implies a stringent evolutionary pressure against protein sub-fragments that can independently fold into stable structures. The symmetric self-association between two identical mis-translated CFr sub-domains to generate an extremely stable structure parallels a mechanism for natural protein-fold evolution by modular recombination of protein sub-structures.
Keywords: mistranslation; protein-fold evolution; protein sub-fragments; NMR structure; protein engineering
Abbreviations: AUC, analytical ultra-centrifugation; D2O, deuterium oxide; ESI, electrospray-ionization; MS, mass spectroscopy; CFr, C-terminal fragment; GuHCl, guanidinium hydrochloride; HSQC, heteronuclear single-quantum coherence; NaPi, Sodium phosphate; NOE(SY), nuclear Overhauser effect (spectroscopy); MALDI-TOF, matrix-assisted laser desorption ionization - time of flight; Rg, radius of gyration; RMSD, root-mean-squared deviation; SASA, solvent accessible surface area; SAXS, small-Angle X-ray Scattering; SD, Shine?Dalgarno; TOCSY, total correlation spectroscopy
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