PDBsum entry 2gjh

De novo protein

PDB id: 2gjh

Contents

Protein chains

57 a.a.

PDB id:

2gjh

Name:

De novo protein

Title:

Nmr structure of cfr (c-terminal fragment of computationally designed novel-topology protein top7)

Structure:

Designed protein. Chain: a, b. Engineered: yes

Source:

Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

20 models

Authors:

G.Dantas

Key ref:

G.Dantas et al. (2006). Mis-translation of a computationally designed protein yields an exceptionally stable homodimer: implications for protein engineering and evolution. J Mol Biol, 362, 1004-1024. PubMed id: 16949611 DOI: 10.1016/j.jmb.2006.07.092

Date:

30-Mar-06 Release date: 17-Oct-06

Protein chains

No UniProt id for this chain

Struc: 57 a.a.

DOI no: 10.1016/j.jmb.2006.07.092

J Mol Biol 362:1004-1024 (2006)

PubMed id: 16949611

Mis-translation of a computationally designed protein yields an exceptionally stable homodimer: implications for protein engineering and evolution.

G.Dantas, A.L.Watters, B.M.Lunde, Z.M.Eletr, N.G.Isern, T.Roseman, J.Lipfert, S.Doniach, M.Tompa, B.Kuhlman, B.L.Stoddard, G.Varani, D.Baker.

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 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.

Selected figure(s)

Figure 7.
Figure 7. Comparison of the Top7 and CFr structures. (a) and (b) Ribbon diagrams of residues 3–51 from one subunit of the CFr NMR structure (green) superimposed on the corresponding region of the Top7 X-ray structure (purple). The backbone RMSD value over these residues is 1.12 Å. The two diagrams are related by a 90° rotation around the vertical axis in the plane of the page.

Figure 9.
Figure 9. Backbone Dynamics of CFr. (a) ^15N T[1] measurements; (b) ^15N T[2] measurements; (c) ^15N HetNOE measurements.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 362, 1004-1024) copyright 2006.

Figures were selected by an automated process.