De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy

Huang, Po-Ssu; Feldmeier, Kaspar; Parmeggiani, Fabio; Velasco, Daniel Alejandro Fernández; Höcker, Birte; Baker, David

doi:10.1038/nchembio.1966

Cited by 236 publications

(234 citation statements)

References 46 publications

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“…[1][2][3] The formation of coiled coils from the assembly of α-helices was first predicted over 60 years ago for naturally occurring proteins and peptides. 4 De novo peptides that fold into coiled coils have proven to be a robust scaffold with interesting properties.…”

Section: Introductionmentioning

confidence: 99%

X-ray Crystallographic Structure and Solution Behavior of an Antiparallel Coiled-Coil Hexamer Formed by de Novo Peptides

Spencer

Hochbaum

2016

Biochemistry

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The self-assembly of peptides and proteins into higher-ordered structures is encoded in the amino acid sequence of each peptide or protein. Understanding the relationship among the amino acid sequence, the assembly dynamics, and the structure of well-defined peptide oligomers expands the synthetic toolbox for these structures. Here, we present the X-ray crystallographic structure and solution behavior of de novo peptides that form antiparallel coiled-coil hexamers (ACC-Hex) by an interaction motif neither found in nature nor predicted by existing peptide design software. The 1.70 Å X-ray crystallographic structure of peptide 1a shows six α-helices associating in an antiparallel arrangement around a central axis comprising hydrophobic and aromatic residues. Size-exclusion chromatography studies suggest that peptides 1 form stable oligomers in solution, and circular dichroism experiments show that peptides 1 are stable to relatively high temperatures. Small-angle X-ray scattering studies of the solution behavior of peptide 1a indicate an equilibrium of dimers, hexamers, and larger aggregates in solution. The structures presented here represent a new motif of biomolecular self-assembly not previously observed for de novo peptides and suggest supramolecular design principles for material scaffolds based on coiled-coil motifs containing aromatic residues.

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Section: Introductionmentioning

confidence: 99%

X-ray Crystallographic Structure and Solution Behavior of an Antiparallel Coiled-Coil Hexamer Formed by de Novo Peptides

Spencer

Hochbaum

2016

Biochemistry

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“…Also, small and less regular structures have been designed by non-computational consensus approaches and fragment assembly [8,9]. However, designing larger (>70 aa) globular αβ proteins with irregular contact patterns is a highly complex task and has only been achieved by employing computational methods [10–14]. In addition to this unique achievement, computational methods have been employed to full-sequence design of a variety of protein structures including early mini-proteins [15–17], tandem repeats [18,19], and ligand binders [20–21].…”

Section: Introductionmentioning

confidence: 99%

“…In addition to this unique achievement, computational methods have been employed to full-sequence design of a variety of protein structures including early mini-proteins [15–17], tandem repeats [18,19], and ligand binders [20–21]. Despite much effort, however, the total number of full-sequence designed proteins for which an atomic resolution structure has been solved still remains low; to our knowledge, less than 10 larger globular αβ proteins have been reported in the literature [10,12–14,22]. Among the available computational methods used here, Rosetta is by far the best validated, and thus, we base this study on the Rosetta software.…”

Section: Introductionmentioning

confidence: 99%

Computational Redesign of Thioredoxin Is Hypersensitive toward Minor Conformational Changes in the Backbone Template

Johansson

Johansen

Christensen

et al. 2016

Journal of Molecular Biology

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Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose six sequences from each of the eight templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to (1) produce soluble protein in Escherichia coli and (2) yield stable monomeric protein, and (3) on the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference among the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when optimizing the geometry of a structure prior to computational design when using a method that is based on rigid conformations.

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“…This paradigm has led to impressive advances, particularly in designing proteins of around 100 amino acids or less (Koga et al, 2012;Kuhlman et al, 2003), and most recently with a full TIM barrel protein thanks to the internal structural symmetry displayed by this fold (Huang et al, 2016). The sTIM of Huang et al has been designed using an approach different to that used by us for Octarellin V: they have considered a quarter of the protein and then, thanks to the symmetry, replicated it in the space to get the full protein, instead of a full idealized backbone as we described for our protein back in 2003.…”

Section: Discussionmentioning

confidence: 99%

“…The few successes reported so far in the construction of large artificial proteins often involved assembly of multiple copies of the same motif, each not exceeding 40 amino acids in length (Parmeggiani et al, 2008;Urvoas et al, 2010). Among the last group, clearly protrudes from the rest the work of Huang et al (2016), where they clearly succeeded in the design, production and characterization of an artificial TIM barrel protein of 184 amino acids, taking advantage of the structural internal symmetry of the protein, repeating four times the same motif.…”

Section: Introductionmentioning

confidence: 99%

The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools

Figueroa¹,

Sleutel²,

Vandevenne³

et al. 2016

Journal of Structural Biology

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a b s t r a c tDespite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (aba) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10 years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features.

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De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy

Cited by 236 publications

References 46 publications

X-ray Crystallographic Structure and Solution Behavior of an Antiparallel Coiled-Coil Hexamer Formed by de Novo Peptides

X-ray Crystallographic Structure and Solution Behavior of an Antiparallel Coiled-Coil Hexamer Formed by de Novo Peptides

Computational Redesign of Thioredoxin Is Hypersensitive toward Minor Conformational Changes in the Backbone Template

The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools

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