Contributions of the Complementarity Determining Regions to the Thermal Stability of a Single-Domain Antibody

<div><p>Single domain antibodies (sdAbs) are the recombinantly-expressed variable domain from camelid (or shark) heavy chain only antibodies and provide rugged recognition elements. Many sdAbs possess excellent affinity and specificity; most refold and are able to bind antigen after thermal denaturation. The sdAb A3, specific for the toxin Staphylococcal enterotoxin B (SEB), shows both sub-nanomolar affinity for its cognate antigen (0.14 nM) and an unusually high melting point of 85°C. Understanding the source of sdAb A3’s high melting temperature could provide a route for engineering improved melting temperatures into other sdAbs. The goal of this work was to determine how much of sdAb A3’s stability is derived from its complementarity determining regions (CDRs) versus its framework. Towards answering this question we constructed a series of CDR swap mutants in which the CDRs from unrelated sdAbs were integrated into A3’s framework and where A3’s CDRs were integrated into the framework of the other sdAbs. All three CDRs from A3 were moved to the frameworks of sdAb D1 (a ricin binder that melts at 50°C) and the anti-ricin sdAb C8 (melting point of 60°C). Similarly, the CDRs from sdAb D1 and sdAb C8 were moved to the sdAb A3 framework. In addition individual CDRs of sdAb A3 and sdAb D1 were swapped. Melting temperature and binding ability were assessed for each of the CDR-exchange mutants. This work showed that CDR2 plays a critical role in sdAb A3’s binding and stability. Overall, results from the CDR swaps indicate CDR interactions play a major role in the protein stability.</p> </div

Effect of Linker Length on Cell Capture by Poly(ethylene glycol)-Immobilized Antimicrobial Peptides

Development of antimicrobial peptide (AMP)-functionalized materials has renewed interest in using poly­(ethylene glycol) (PEG)-mediated linking to minimize unwanted interactions while engendering the peptides with sufficient flexibility and freedom of movement to interact with the targeted cell types. While PEG-based linkers have been used in many AMP-based materials, the role of the tether length has been minimally explored. Here, we assess the impact of varying the length of PEG-based linkers on the binding of bacterial cells by surface-immobilized AMPs. While higher surface densities of immobilized AMPs were observed using shorter PEG linkers, the increased density was insufficient to fully account for the increased binding activity of peptides. Furthermore, effects were specific to both the peptide and cell type tested. These results suggest that simple alterations in linking strategiessuch as changing tether lengthmay result in large differences in the surface properties of the immobilized AMPs that are not easily predictable

Primary structure and sequence of sdAbs used in this study.

<p>A) The overall primary structure of sdAbs is shown schematically with alternating framework and CDRs. Melting temperature for the wildtype sdAbs is given in parentheses next to the name. The framework regions are grouped together above the schematic while the CDRs are shown below. The percent identity of sdAb D1 and sdAb C8 toward sdAb A3 is shown for each region. B) Construct identifications are shown schematically for all hybrid antibodies in this study. Regions are color coded for clarity. Observed melting point is shown as a bar graph. Detailed measurements are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077678#pone-0077678-t001" target="_blank">Table 1</a>.</p

CDR swaps lower affinity but may increase or decrease melting temperature.

<p>Melting temperature for sdAbs A3, D1 and C8, as well as full CDR swaps, are plotted against affinity. For each data point the framework origin is indicated before the dot while the CDR origin is indicated after the dot. The affinity shown is that towards the target specified for the CDR origin antibody.</p

Locations of the disulfide bonds added to sdAb A3 in Constructs 1 through 4.

<p>The amino acid sequence of sdAb A3 is given with lines connecting the residues which are changed to cysteine in the constructs as shown. The three dimensional location of each new disulfide bond is shown as an orange-colored space-filling representation of the amino acid pair in the crystal structure of sdAb A3. The CDRs are color coded blue, green, and red, respectively. The native disulfide bond is shown as a standard color space-filling representation.</p

Melting temperatures and affinities.

a<p>Wild type sdAb A3 contains a disulfide bond between cysteines at positions 22 and 99 of the sdAb. Constructs with cysteines at those positions (C22/C99) are listed as "yes".</p><p>Melting temperatures and affinities.</p

Sequences of L1 binding sdAb.

<p>The alignment shows the sequences of L1 binders isolated in sandwich-style selection. Three families, based on homology within the CDR regions were isolated. CDR regions are underlined. Red denotes positions where the amino acid is 90% conserved in the compared sequences, blue indicates low consensus (50%) and black are not conserved. The most dominant family (L1-H7 family) showed some variation in amino acid sequences of the framework regions that showed an impact of binding affinities and thermal stability. Arrows mark the positions of the cysteine substitutions in the L1-G2+ construct.</p

Recombinant vaccinia virus proteins and antibodies from BEI resources.

<p>Recombinant vaccinia virus proteins and antibodies from BEI resources.</p

Melting temperatures of vaccinia binding sdAbs.

<p>* Fluorescent melt assays performed in triplicate; the measurements were typically identical within less than 0.2°C, and always within 0.6°C.</p><p>Melting temperatures of vaccinia binding sdAbs.</p

Binding kinetics for sdAbs targeting the L1 antigen^*.

<p>* From binding to at least 3 L1-coated spots, reported as average (standard deviation).</p><p>Binding kinetics for sdAbs targeting the L1 antigen^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106263#nt101" target="_blank">*</a>}.</p