<i>CPQ</i> coding and 3’-UTR SNPs.

<p><i>CPQ</i> coding and 3’-UTR SNPs.</p

Target and linked SNPs for TaqMan assay on intron 6.

<p>“Difference in variant allele frequency” columns are calculated by subtracting the average frequency of the non-reference SNP in susceptible from that in resistant from the GBS data.</p

Scatterplot representing GBS data on chromosome 2.

<p>Difference in SNP frequencies (Y-axis) is plotted between the average non-reference SNP frequencies of the two replicate pools (resistant less susceptible) relative to chromosome 2 position (X-axis) in Mbp. Positive values indicate that non-reference SNP favors resistance, whereas negative values indicate the SNP favors susceptibility for ascites. <b>(Panel 1A):</b> Entire chromosome 2 for males. The black arrow indicates the peak associated with the <i>CPQ</i> gene locus. <b>(Panel 1B):</b> An expanded view of the peak region ((black arrow) from Panel 1A. <b>(Panel 1C):</b> The same region as Panel 1B only for females.</p

Primers, probes, and conditions for RT-qPCR.

<p>For each <i>CPQ</i> locus: position is the [chromosome]:[chromosomal position] of the 5’ base according to the <i>Gallus_gallus-5</i> assembly; Primers are 5’-3’ for forward (F) and reverse (R); Probes are 5’-3’ with allele 1 (P1) labeled with FAM and allele 2 (P2) labeled with HEX. Annealing temperatures for qPCR are indicated in °C.</p

Genotype data for <i>CPQ</i> intron-6 SNPs in broiler lines.

<p>The REL, Y and Z lines were genotyped using 5’-exonuclease assay where TA is homozygous reference allele, YM (TA/CC) is heterozygous, and CC is homozygous non-reference allele. Genotype frequencies (Freq) were determined for the entire line (All) or for the ascites resistant (R) or susceptible (S) subpopulations based on phenotype in a hypobaric challenge. Count for All in each line include genotypes from birds with missing gender. The samples were also analyzed by gender. P-values for a simple Bonferroni correction (Adj Pval) of chi square test of observed vs. expected are presented.</p

Genotype data combining <i>CPQ</i> intron-6 (rs80617053 and rs80618855) and exon-8 (rs738850243) variants for the REL and commercial lines Y and Z.

<p>The genotype designations are indicated within brackets for each pattern. Column and row designations are as for Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189544#pone.0189544.t004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189544#pone.0189544.t005" target="_blank">5</a>. P-values for a simple Bonferroni correction (Adj Pval) of chi square test of observed vs. expected are presented for genotypes with frequency ≥0.10.</p

Full and Partial Agonism of a Designed Enzyme Switch

Chemical biology has long sought to build protein switches for use in molecular diagnostics, imaging, and synthetic biology. The overarching challenge for any type of engineered protein switch is the ability to respond in a selective and predictable manner that caters to the specific environments and time scales needed for the application at hand. We previously described a general method to design switchable proteins, called “chemical rescue of structure”, that builds <i>de novo</i> allosteric control sites directly into a protein’s functional domain. This approach entails first carving out a buried cavity in a protein <i>via</i> mutation, such that the protein’s structure is disrupted and activity is lost. An exogenous ligand is subsequently added to substitute for the atoms that were removed by mutation, restoring the protein’s structure and thus its activity. Here, we begin to ask what principles dictate such switches’ response to different activating ligands. Using a redesigned β-glycosidase enzyme as our model system, we find that the designed effector site is quite malleable and can accommodate both larger and smaller ligands, but that optimal rescue comes only from a ligand that perfectly replaces the deleted atoms. Guided by these principles, we then altered the shape of this cavity by using different cavity-forming mutations, and predicted different ligands that would better complement these new cavities. These findings demonstrate how the protein switch’s response can be tuned <i>via</i> small changes to the ligand with respect to the binding cavity, and ultimately enabled us to design an improved switch. We anticipate that these insights will help enable the design of future systems that tune other aspects of protein activity, whereby, like evolved protein receptors, remolding the effector site can also adjust additional outputs such as substrate selectivity and activation of downstream signaling pathways