Using Engineered Single-Chain Antibodies to Correlate Molecular Binding Properties and Nanoparticle Adhesion Dynamics

Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that bear them

Disordered Domain Shifts the Conformational Ensemble of the Folded Regulatory Domain of the Multidomain Oncoprotein c‑Src

c-Src kinase is a multidomain non-receptor tyrosine kinase that aberrantly phosphorylates several signaling proteins in cancers. Although the structural properties of the regulatory domains (SH3-SH2) and the catalytic kinase domain have been extensively characterized, there is less knowledge about the N-terminal disordered region (SH4UD) and its interactions with the other c-Src domains. Here, we used domain-selective isotopic labeling combined with the small-angle neutron scattering contrast matching technique to study SH4UD interactions with SH3-SH2. Our results show that in the presence of SH4UD, the radius of gyration (Rg) of SH3-SH2 increases, indicating that it has a more extended conformation. Hamiltonian replica exchange molecular dynamics simulations provide a detailed molecular description of the structural changes in SH4UD-SH3-SH2 and show that the regulatory loops of SH3 undergo significant conformational changes in the presence of SH4UD, while SH2 remains largely unchanged. Overall, this study highlights how a disordered region can drive a folded region of a multidomain protein to become flexible, which may be important for allosteric interactions with binding partners. This may help in the design of therapeutic interventions that target the regulatory domains of this important family of kinases

Antigenic <i>Ixodes scapularis</i> salivary proteins identified by immunoscreening a nymphal <i>Ixodes scapularis</i> yeast display library using nymph-immune rabbit serum.

<p>#Homology search performed using BLAST (<a href="http://www.ncbi.nlm.nih.gov/BLAST" target="_blank">www.ncbi.nlm.nih.gov/BLAST</a>).</p><p>*Theoretical molecular weight (MW) and isoelectric point (pI) using ExPASy proteomics server (<a href="http://www.expasy.ch/tools/pi_tool.html" target="_blank">http://www.expasy.ch/tools/pi_tool.html</a>).</p>†<p>Proteins ≤40% identity using the database of VectorBASE (<a href="http://iscapularis.vectorbase.org/" target="_blank">http://iscapularis.vectorbase.org/</a>) and the GenBank database (<a href="http://www.ncbi.nlm.nih.gov/blast/Blast.cgi" target="_blank">www.ncbi.nlm.nih.gov/blast/Blast.cgi</a>).</p>‡<p>Secretory signal sequence as assessed by the SignalP 3.0 signal prediction server (<a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">www.cbs.dtu.dk/services/SignalP/</a>).</p

Purified recombinant <i>Ixodes scapularis</i> salivary proteins.

<p>(A) Coomassie blue staining of purified recombinant <i>I. scapularis</i> salivary proteins rP8, rP19 and rP23 electrophoresed on SDS 12% polyacrylamide gel. (B) Western blot analysis of the recombinant proteins probed with nymph-immune rabbit serum (IRS) and normal rabbit serum (NRS). (C) PAS staining of rP8, rP19, rP23 and Salp15 electrophoresed on SDS 12% polyacrylamide gel.</p

Relationship between post-engorgement weights and resultant sexes in <i>Ixodes scapularis</i> nymphs.

<p>Relationship between post-engorgement weights and resultant sexes in <i>Ixodes scapularis</i> nymphs.</p

Enrichment and selection of yeast cells expressing immunogenic <i>I. scapularis</i> salivary proteins.

<p>FACS analysis of yeast cells using nymph-immune rabbit IgG (red) and IgG derived from normal rabbit serum (blue) of transformed yeast cells (sort 0); autoMACS sort (AutoMACS); MidiMACS sort 1, 2 and 3 (MidiMACS1, 2 and 3). (B) Selection for IgG binding clones using 50 µg/ml Alexa-488 conjugated nymph immune rabbit IgG. Sort 0 was used as a negative control and MACS sort 4 as a positive control. Upper panel: mouse anti-Xpress antibody binding to induced yeast cells (PE). Lower panel: Cells binding nymph immune rabbit IgG (IR IgG) shown within the pink gate (Alexa-488). (C) Titration of binding of each unique using 50 µg/ml (green bars), 10 µg/ml (red bars) or 1 µg/ml (blue bars) of immune rabbit IgG. The percentage IgG binding yeast cells were determined by FACS analysis as was shown in the right lower panel of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015926#pone-0015926-g001" target="_blank">Fig. 1B</a>.</p

Primers used.

<p>Primers used.</p

Influence of recombinant salivary proteins on human coagulation system.

<p>(A) Thrombin generation was initiated in human pooled normal plasma with 1 pM tissue factor (TF) in the presence of rP8 (orange), rP19 (green) or rP23 (red) and thrombin generation was measured using a fluorogenic substrate. (B) Lagtime, (C) time to peak (ttpeak) and (D) Endogenous Thrombin Potential (ETP) were measured. Unpaired t-test was used to determine statistical significance. Representatives of three experiments are shown. Results described represent the mean ± SEM.</p

Influence of recombinant salivary proteins on human complement system.

<p>Serum sensitive strain <i>Borrelia garinii</i> A87S was incubated with 12.5% NHS in the presence of BSA, Salp15, rP19, rP23 or rP8 for (A) 1.5 hours; or (B) 4.5 hours and the percentage of immotile spirochetes were determined. Control spirochetes were incubated with heat-inactivated NHS (HI). Two hundred spirochetes were counted. Results represent mean ± SEM of values from a representative of 3 replicate experiments.</p

Nymph feeding after rabbit immunization with recombinant salivary proteins.

<p>(A) Nymph salivary gland extract probed with rP8/rP19/rP23 immune rabbit serum (panel 1), rP8 or rP19 or rP23 immune rabbit serum (panel 2), and with serum from the control (OVA) rabbit. (B) Nymph weights recovered from the control and the rP8/rP19/rP23 immunized rabbits. Tick weights of the heavy group of nymphs (C) and light group of nymphs (D) fed on rP8/rP19/rP23 immunized rabbits compared to the control rabbits. Weights of the heavy group of nymphs (E) and light group (F) of nymphs fed on rabbits immunized singly with OVA, rP19, rP8 or rP23 respectively. The horizontal bars represent the means of the respective groups. Unpaired t-test was used to determine statistical significance.</p