Protein sequences of parental and graft variants.

<p>Protein sequence of DSTL096, the SP-15 stable sdAb framework donor, and the two graft mutants are shown in the top sequence comparison. Conserved sequences are shown in gray, and differences in black. The CDR regions are indicated by colored bars (orange for CDR1, green for HV2, and blue for CDR3). The region designated as HV4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160534#pone.0160534.ref015" target="_blank">15</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160534#pone.0160534.ref040" target="_blank">40</a>] is indicated by a gray bar, and was considered as framework for this work. The natural canonical and non-canonical disulfide bonds are indicated by lines joining the cysteines, with the canonical in red and non-canonical in purple. The bottom panel shows a close up of the area containing the three positions that differ between SP15-096-13 and SP15-096-123. Point mutations were constructed to produce the six intermediate proteins.</p

Characterization of DSTL096, DSTL097, shark096 and original graft constructs.

<p>Characterization of DSTL096, DSTL097, shark096 and original graft constructs.</p

Characterization of point mutants.

<p>Characterization of point mutants.</p

Schematic of the shark096 sdAb structure.

<p>Two views of a nurse shark sdAb structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160534#pone.0160534.ref036" target="_blank">36</a>] (PDB #1sq2) with high sequence homology to shark096. The CDR regions are color coded red, green, blue for CDRs 1 2 and 3 respectively and disulfide bonds are in yellow. In ball and stick are shown the position of the three amino acids that differed between the two graft structures (SP15-096-123 and SP15-096-13). The third panel shows the specific residues for clarity. The sequence of shark096 is shown and that for 1sq2 is shown as a difference plot. Dots indicate identical residues. The disulfide bonds are indicated by lines and the three mutated residues are identified by overscores. Although the CDR2 region is truncated and presumably has no contact with antigen, it was found critical for both stability and affinity.</p