Summary of experimental validation results for the five designed sequences^a.

a<p>The sign of “+” and “−” indicates positive and negative experimental results respectively.</p>b<p>RMSD between the first I-TASSER model and the target scaffold.</p>c<p>Protein expression and solubility determined by visual identification via comassie stain gels.</p>d<p>Presence of secondary structural elements defined by circular dichroism.</p>e<p>Possession of a stable tertiary fold determined by the presence of secondary structural elements (CD) and NMR spectroscopy.</p>f<p>Percentage of α-helix residues decided by the CD spectra (the values in parentheses are the number in the scaffold structure.</p

Evaluation of designed sequences.

<p>Data is averaged over 87 test proteins. The details on each protein can be found at <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003298#pcbi.1003298.s007" target="_blank">Table S2</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003298#pcbi.1003298.s008" target="_blank">S3</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003298#pcbi.1003298.s009" target="_blank">S4</a>.</p>a<p>TM-score between the first I-TASSER model and the target scaffold.</p>b<p>RMSD between the first I-TASSER model and the target scaffold.</p>c<p>SS: Secondary structure.</p>d<p>SA: Solvent accessibility.</p>e<p>PBM: Physics-based method using FoldX.</p>f<p>EvBM: Evolution-based method using only evolutionary terms in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003298#pcbi.1003298.e004" target="_blank">Eq. (1)</a>.</p>g<p>EBM: Evolutionary based method using both evolutionary and physics-based terms in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003298#pcbi.1003298.e006" target="_blank">Eq. (3)</a>.</p

An Evolution-Based Approach to <i>De Novo</i> Protein Design and Case Study on <i>Mycobacterium tuberculosis</i>

<div><p>Computational protein design is a reverse procedure of protein folding and structure prediction, where constructing structures from evolutionarily related proteins has been demonstrated to be the most reliable method for protein 3-dimensional structure prediction. Following this spirit, we developed a novel method to design new protein sequences based on evolutionarily related protein families. For a given target structure, a set of proteins having similar fold are identified from the PDB library by structural alignments. A structural profile is then constructed from the protein templates and used to guide the conformational search of amino acid sequence space, where physicochemical packing is accommodated by single-sequence based solvation, torsion angle, and secondary structure predictions. The method was tested on a computational folding experiment based on a large set of 87 protein structures covering different fold classes, which showed that the evolution-based design significantly enhances the foldability and biological functionality of the designed sequences compared to the traditional physics-based force field methods. Without using homologous proteins, the designed sequences can be folded with an average root-mean-square-deviation of 2.1 Å to the target. As a case study, the method is extended to redesign all 243 structurally resolved proteins in the pathogenic bacteria <i>Mycobacterium tuberculosis</i>, which is the second leading cause of death from infectious disease. On a smaller scale, five sequences were randomly selected from the design pool and subjected to experimental validation. The results showed that all the designed proteins are soluble with distinct secondary structure and three have well ordered tertiary structure, as demonstrated by circular dichroism and NMR spectroscopy. Together, these results demonstrate a new avenue in computational protein design that uses knowledge of evolutionary conservation from protein structural families to engineer new protein molecules of improved fold stability and biological functionality.</p></div

Average fractional difference in amino acid composition between the target and designed sequences.

<p>(A) EBM; (B) PBM.</p

Average RMSD between the scaffold structures and I-TASSER models on the proteins designed by PBM and EBM.

<p>The dataset is divided by the TM-score cutoff of the template proteins that were used for constructing the sequence profiles.</p

Illustration of protein design on the soluble human CD59.

<p>(A) X-ray structure of the target protein. (B) I-TASSER model on the EBM designed sequence. (C) I-TASSER model of the PBM designed sequence. (D) Secondary structure of the target assigned by DSSP, in comparison to that predicted by PSSpred on the target (PSSPred_WT), the EBM (PSSPred_EBM), and the PBM designed sequences (PSSPred_PBM). ‘E’ stands for sheet, ‘H’ for helix and ‘C’ for coil.</p

Circular dichroism and ¹H 1D NMR spectrum of designed proteins.

<p>(A, F) hnRNPK; (B, G) thioredoxin; (C, H) CISK-PX; (D, I) Lov2; (E, J) TIF1 domains. The first column represents CD data, with X-axis representing the wavelength of circular polarized light (nm) and Y-axis the mean residue ellipticity measured in degree cm² dmol⁻¹. The second column consists of ¹H 1D NMR spectra, with chemical shifts given in ppm on the X-axis. The arrows indicate key methyl chemical shifts indicated of a stable protein fold.</p

Illustrative examples of the EBM design on <i>M. tuberculosis</i> proteins.

<p>(A) Superposition of I-TASSER model of the EBM sequence (green) on the target structure from the thioredoxin C (red) with a RMSD 2.52 Å. (B) Sulfate ion binding with the designed protein where ion-protein hydrogen bonds are highlighted by dashed lines. (C) The EBM binding site analogous position on the target indicates that it cannot accommodate the sulfate ion (dotted sphere) due to steric overlaps. (D) Superposition of the PZAase protein (red) and the I-TASSER model on the EBM sequence (green) with a RMSD 0.28 Å. (E) Active site residues of PZAase as represented in sticks. Triad (green) and Zn²⁺ binding sites (red) are retained in the designed protein. Gray color indicates mutations at the active site. (F) Binding pockets identified by COFACTOR with red spacefill indicating an isochorismic acid binding site and blue the sulfate ion binding site. Y132I mutation in EBM design is designated by yellow. The figure was generated using Pymol and Adobe Photoshop software.</p

Free energy of folding was determined by circular dichroism.

<p>(A) thioredoxin; (B) CISK-PX domains. The figure plots free energy (kJ/mol) versus the concentration [M] of chemical denaturant urea. The unfolding assay was conducted in 25 mM NaPO₄, 150 mM NaF, pH 7.5 with 2–3 uM protein concentration and 0–9.5 M urea concentrations at 298 K. The free energies of folding are equal to the intercept through linear regression <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003298#pcbi.1003298-Greenfield1" target="_blank">[76]</a>.</p

Results of I-TASSER folding on 45 sequences from previous protein design experiments [18], [39]–[51].

<p>(A, B) Estimated TM-score and RMSD of the I-TASSER predicted models versus the actual TM-score and RMSD of the models to the experimental structure for the 16 folded designs. The estimation is calculated based on C-score with an error bar obtained from large-scale benchmark data <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003298#pcbi.1003298-Zhang7" target="_blank">[52]</a>. (C, D, E) Histogram distributions of estimated TM-score and RMSD, and C-score of the I-TASSER predictions for 16 folded sequences (open circles and solid lines) and 29 unfolded sequences (stars and dashed lines). The vertical lines mark the average values for the folded and unfolded sequences respectively.</p