Directed Assembly of Elastic Fibers via Coacervate Droplet Deposition on Electrospun Templates.

Elastic fibers provide critical elasticity to the arteries, lungs, and other organs. Elastic fiber assembly is a process where soluble tropoelastin is coacervated into liquid droplets, cross-linked, and deposited onto and into microfibrils. While much progress has been made in understanding the biology of this process, questions remain regarding the timing of interactions during assembly. Furthermore, it is unclear to what extent fibrous templates are needed to guide coacervate droplets into the correct architecture. The organization and shaping of coacervate droplets onto a fiber template have never been previously modeled or employed as a strategy for shaping elastin fiber materials. Using an in vitro system consisting of elastin-like polypeptides (ELPs), genipin cross-linker, electrospun polylactic-co-glycolic acid (PLGA) fibers, and tannic acid surface coatings for fibers, we explored ELP coacervation, cross-linking, and deposition onto fiber templates. We demonstrate that integration of coacervate droplets into a fibrous template is primarily influenced by two factors: (1) the balance of coacervation and cross-linking and (2) the surface energy of the fiber templates. The success of this integration affects the mechanical properties of the final fiber network. Our resulting membrane materials exhibit highly tunable morphologies and a range of elastic moduli (0.8-1.6 MPa) comparable to native elastic fibers.

Structural Conformation and Activity of Spider-Derived Inhibitory Cystine Knot Peptide Pn3a Are Modulated by pH.

Numerous spider venom-derived gating modifier toxins exhibit conformational heterogeneity during purification by reversed-phase high-performance liquid chromatography (RP-HPLC). This conformational exchange is especially peculiar for peptides containing an inhibitor cystine knot motif, which confers excellent structural stability under conditions that are not conducive to disulfide shuffling. This phenomenon is often attributed to proline cis/trans isomerization but has also been observed in peptides that do not contain a proline residue. Pn3a is one such peptide forming two chromatographically distinguishable peaks that readily interconvert following the purification of either conformer. The nature of this exchange was previously uncharacterized due to the fast rate of conversion in solution, making isolation of the conformers impossible. In the present study, an N-terminal modification of Pn3a enabled the isolation of the individual conformers, allowing activity assays to be conducted on the individual conformers using electrophysiology. The conformers were analyzed separately by nuclear magnetic resonance spectroscopy (NMR) to study their structural differences. RP-HPLC and NMR were used to study the mechanism of exchange. The later-eluting conformer was the active conformer with a rigid structure that corresponds to the published structure of Pn3a, while NMR analysis revealed the earlier-eluting conformer to be inactive and disordered. The exchange was found to be pH-dependent, arising in acidic solutions, possibly due to reversible disruption and formation of intramolecular salt bridges. This study reveals the nature of non-proline conformational exchange observed in Pn3a and possibly other disulfide-rich peptides, highlighting that the structure and activity of some disulfide-stabilized peptides can be dramatically susceptible to disruption.

Changes in Potency and Subtype Selectivity of Bivalent NaV Toxins are Knot-Specific.

Disulfide-rich peptide toxins have long been studied for their ability to inhibit voltage-gated sodium channel subtype NaV1.7, a validated target for the treatment of pain. In this study, we sought to combine the pore blocking activity of conotoxins with the gating modifier activity of spider toxins to design new bivalent inhibitors of NaV1.7 with improved potency and selectivity. To do this, we created an array of heterodimeric toxins designed to target human NaV1.7 by ligating a conotoxin to a spider toxin and assessed the potency and selectivity of the resulting bivalent toxins. A series of spider-derived gating modifier toxins (GpTx-1, ProTx-II, gHwTx-IV, JzTx-V, CcoTx-1, and Pn3a) and two pore-blocker µ-conotoxins, SxIIIC and KIIIA, were used for this study. We employed either enzymatic ligation with sortase A for C- to N-terminal ligation or click chemistry for N- to N-terminal ligation. The bivalent peptide resulting from ligation of ProTx-II and SxIIIC (Pro[LPATG6]Sx) was shown to be the best combination as native ProTx-II potency at hNaV1.7 was conserved following ligation. At hNaV1.4, a synergistic effect between the pore blocker and gating modifier toxin moieties was observed, resulting in altered sodium channel subtype selectivity compared to the parent peptides. Further studies including mutant bivalent peptides and mutant hNaV1.7 channels suggested that gating modifier toxins have a greater contribution to the potency of the bivalent peptides than pore blockers. This study delineated potential benefits and drawbacks of designing pharmacological hybrid peptides targeting hNaV1.7.