In silico and in vivo combinatorial design of Octarellin VI, an artificial protein modeled on the (B/A)8 fold

One way to gain insight into the sequence-structure-function relationship in proteins is to perform de novo design of artificial proteins. The applications of such a study are varied. For example, in medicine and industry, it would give us the ability to precisely engineer proteins to perform a specific function under a wider range of conditions. Despite impressive successes in the de novo protein design, designing a folded protein of more than 100 amino acids remains a challenge. In our lab, four generations of Octarellins, de novo polypeptides of more than two hundred amino acids modelled on the (beta/alpha)8 barrel fold, have been built and structurally characterized using biophysical and spectroscopic methods. The last generation of Octarellins was designed following a hierarchical method combining the specificity of rational design and the power of computational design. The resulting artificial protein, named Octarellin VI, was expressed in E. coli and purified from inclusion bodies. The biophysical characterization showed a monomeric protein, with a secondary structure level similar to the computationally designed model and thermostability. However, the poor solubility in bacteria and low stability of the protein at long term make impossible determine its structure to criticize the model. To improve these negative features, we performed a directed evolution process over the Octarellin, following the improvement at solubility level in the bacteria, thanks to the fusion of Octarellin to the fluorescent folding reporter GFP. After 8 cycles of directed evolution by Error Prone PCR technique, we obtained a most soluble protein, with a 92% of sequence identity with the original protein. This soluble variant is under study to characterize its structural features. The combination between in silico design and directed evolution process emerges as a powerful tool for protein engineering, showing be complementaries techniques and the information obtained by the whole process of design and posterior comparison between 3D structure of Octarellin with the computational model will allow to improve the algorithms for protein design

A novel protocol for the design of artificial (β/α)8-barrel proteins

Designing de novo proteins of more than 100 amino acids is still challenging. The creation of artificial (β/α)8-barrel proteins had only one successful example in literature, thank to use of internal spatial symmetry. Here we present a protocol to design de novo (β/α)8-barrel proteins without symmetry restriction. First, the backbone was created in 4 steps: (I) Rosetta ParametricDesign produced an highly symmetric polyalanine scaffold with no loops; (II) Rosetta Fixed-Backbone Design used the previous output to substitute the alanines in all the position; (III) Loops were constructed with Modeller joining the terminus of the secondary structure elements and (IV) RosettaRelax performed relaxation, creating around 4000 different models. 28 backbone models were selected for the next steps of sequence design. To design the final proteins for experimental validation, 10 cycles of Rosetta Design and Relax were performed. In the first cycle only apolar amino acids were allowed in hydrophobic regions; in the next 6 cycles, amino acids were allowed based on the definition of 3 regions: core, boundaries and surface. All the amino acids were allowed in each position in the last 3 cycles. More than 10000 different sequences were created and analyzed in term of amino acid composition, sequence similarity with natural protein, secondary structure prediction, and molecular dynamics simulations. The 30 best candidate sequences have been selected for experimental verification.Octarellin VI

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

Despite 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 (alphabetaalpha) 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 10years 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