Effective number of substitutions versus distance to the active site and solvent exposure.

<p>Effective number of amino acids that appear at each position (<i>k</i>*) plotted versus the distance from its Cα atom to the active site (A) and against the fraction of amino acid surface exposed to the solvent (B). The position of the active site was defined as the average position of the Ser70, Lys73 and Glu166 Cα atoms; surface exposure was computed with the POPS webserver [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118684#pone.0118684.ref048" target="_blank">48</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118684#pone.0118684.ref049" target="_blank">49</a>] based on the X-ray structure deposited under PDB entry 1XPB [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118684#pone.0118684.ref050" target="_blank">50</a>]. Dot colors correspond to correlations against the most often matched properties: blue for <i>volume</i>, cyan for <i>volume/(P(helix)+P(sheet))</i>, green for <i>steric hindrance</i>, dark green for <i>steric hindrance / P(sheet)</i>, red for <i>hydrophobicity</i>, magenta for <i>log(solubility) x hydrophobicity</i>, orange for <i>flexibility x hydrophobicity</i>. Black dots represent correlations with FoldX predictions. Gray dots correspond to the rest of the residues (with other or no detected correlations). The line drawn in panel A arbitrarily indicates how the maximum possible <i>k</i>* increases with distance to the active site.</p

Examples of correlations detected between ΔΔG^stat values and amino acid descriptors (a-r) or ΔΔG^FoldX (s-t).

<p>The gray circles point at the wild type amino acid, gray squares point at substitutions that have been observed in natural TEM variants. Lines correspond to best linear fits.</p

Structural representation of the (a) Leu51 (b) Asn170 (c) Arg222 regions in the TEM-1 β-lactamase structure.

<p>Pictures rendered from PDB ID 1XPB [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118684#pone.0118684.ref050" target="_blank">50</a>] using the program PyMOL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118684#pone.0118684.ref051" target="_blank">51</a>]. Atom colors are red for oxygen, blue for nitrogen, gray for carbon and yellow for hydrogen.</p

Structure mapping of the residues whose substitution patterns can be explained by the nine most common descriptors.

<p>The mapped amino acids are shown as red spheres, and residues Ser70, Lys73 and Glu166 as green spheres. All residue representations lack the main chain nitrogen, carbonyl carbon and oxygen atoms for clarity. The letters on the bottom right of each panel indicate the wild type amino acids most often found at the indicated locations, with the font size being roughly proportional to the relative number of occurrences of the amino acid. The small bar on the bottom right of each panel measures the fractional solvent exposure of the wild type residues to which the descriptor was mapped.</p

Protein-DNA interactions of potentially improved RVD loops for targeting guanine.

<p>(<b>A</b>) Wild type (N12-N13) alone (grey) and superposed to: (<b>B</b>) N12-N13K mutant (yellow), (<b>C</b>) N13K-G14* mutant (green) and (<b>D</b>) N13*-G14K mutant (magenta).</p

Structural and energetic features of TAL-DNA interaction.

<p>(<b>A</b>) Mean residue fluctuation (RMSD) computed for the DNA-bound and <i>apo</i> states of the 11.5-repeats TAL system (TAL[11.5]/P1 and TAL[11.5]/P1-apo); averages are performed over all the repeats; bars represent standard deviations. The same trend is observed for all simulated systems (<i>cf. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080261#pone.0080261.s005" target="_blank">Figure S5</a>). (<b>B</b>) Contribution to the total DNA-binding energy from different sections of TAL subdivided by type and calculated on the DNA-bound 22.5-repeat TAL system (TAL[22.5]/P1) using MM/GBSA (Number of residues contributing to each type: G13 = 6, N13 = 2, I13 = 7, D13 = 5, G14/K16/Q17 = 20, others = 600, N-terminus = 97). Repeats containing a deletion at position X13 have been excluded from the statistics. (<b>C</b>) Per repeat mean energy contribution to the total DNA-binding energy; averages are performed over all the repeats of the DNA-bound 22.5-repeat TAL system (TAL[22.5]/P1). Repeats containing a deletion at X13 position have been excluded from the statistics. The complete binding energy profile is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080261#pone.0080261.s009" target="_blank">Figure S9</a>.</p

Simulation details for each system.

<p>Simulation details for each system.</p

Protein-DNA binding energies for modified targets of repeat 7 (N*) of the PthXo1 system (TAL[22.5]/P1).

<p>The base pairs corresponding to each category are: wild type (CG), mutated (TA) and 5-methylcytosine (mCG). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080261#pone.0080261.s013" target="_blank">Methods S1</a> for details.</p

Schematic representation of the TAL-DNA interface.

<p>(<b>A</b>) Cartoon representation of one single repeat interacting with DNA. The <i>oxyanion clip</i> (G14, K16 and Q17) interacts with the phosphate group of the <i>(i-1)^th</i> base, thus fixing the position of the X13 side-chain with respect to the <i>i^th</i> base and freezing its structural fluctuation. Dashed circles indicate the interaction radii of different X13 residues, sorted by side-chain size: the inner circle corresponds to G13/*13, while the others are represented by the outer circle. Only loci α, β and γ are sampled by the side-chains of X13, resulting in incomplete molecular differentiation. (<b>B</b>) Pharmacophore-like model for the nucleobases discussed in the text (left). Dots represent sites with variable properties across nucleobases; colours are used to highlight the characteristics of the substituents: green for pyridine-like (H-bond acceptor) nitrogen atoms, blue for pyrrole-like and amine (H-bond donor) nitrogen atoms, grey for methyl groups, and red for carbonyl oxygen atoms. On the right, relative molecular electrostatic potential (MEP) maps for the corresponding nucleobases are reported. Calculations were performed at the QM level on methyl-capped purines (N9) and pyrimidines (N1) (red  =  −5.0 k_BT/e, blue  =  5.0 k_BT/e, isovalue 4.0 E⁻⁴).</p

TAL repeats architecture.

<p>(<b>A</b>) Top view of bound TAL along the DNA axis showing the N-terminus (structure from 3UGM). (<b>B</b>) Side view of bound TAL displayed from the N-terminus (bottom) to the C-terminus (top) (structure from 3UGM). (<b>C</b>) TAL without DNA and the RVDs explicitly depicted (laying on the inner-side of the super-helix). The orange line represents the DNA axis. The protein is orientated from N-terminus (bottom) to C-terminus (top). A larger pitch compared to the bound structure is clearly observable (structure from 3V6P). Protein: grey; DNA: orange.</p