HVDC Transmission: A Path to the Future?

Direct current transmission has been the poor stepchild of the U.S. electric industry. Although early-generation plants were based on DC technology, it was soon deemed uneconomical to transmit electricity over long distances, but it now appears poised for a change. Both the increasing technical potential and changing economics of HVDC lines promise a growing role in the future.

Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein.

Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity

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

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

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 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