A 27 amino acid collagen-based peptide (Hbyp3) was designed to radially display nine hydrophobic bipyridine moieties from a triple helical scaffold. Self-assembly of such functionalized triple helices led to the formation of micrometer-scaled disks with a curved morphology, presumably mediated by aromatic interactions, with a height that is in the range of the length of the triple helical peptide. Higher order assembly of these curved disks into micrometer-sized hollow spheres was accomplished through metal-ligand interactions between bipyridine groups of the disks and metal ions such as Fe(II), Co(II), Zn(II) and Cu(II). The thickness of the shell of these hollow spheres corresponds well with the thickness of the collagen peptide-based triple helix and the corresponding self-assembled disks. Addition of a metal ion chelator was found to reverse the assembly of the hollow spheres back to the curved disk structures. These data support the formation of the hollow spheres from the self-assembled disks of Hbyp3 upon addition of metal ions.

The hierarchical assembly of collagen-based triple helical peptides into disks, followed by metal-promoted assembly into microcages is described. The length of the triple helix was found to correlate to the height of the disks that formed, providing mechanistic insights into their formation. The encapsulation of fluorescently labeled dextrans within the peptide microcages, and their subsequent thermal release is detailed. The half-life for thermal release of the encapsulated cargo from the cages was found to increase as the peptide triple helix stability increased, providing a means to engineer cargo delivery rates through peptide design.

Insulin-signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to structural reorganization. To test the functional coupling between insulin’s “hinge opening” and receptor activation, we inserted an artificial ligand-dependent switch into the insulin molecule. Ligand-binding disrupts an internal tether designed to stabilize the hormone’s native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (meta-fluoro-phenylboronic acid at GlyA1) and internal diol (3,4-dihydroxybenzoate at LysB28). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin-signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the hormone. Similarly, metabolite-regulated signaling was not observed in control studies of 1) an unmodified insulin analog or 2) an analog containing a diol sensor without internal tethering. Although secondary structure (as probed by circular dichroism) was unaffected by ligand-binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and allosteric holoreceptor signaling. In addition to this foundational finding, our results provide proof of principle for design of a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a “smart” insulin analog to mitigate hypoglycemic risk in diabetes therapy.

We optimized a peptide extraction and LC–MS protocol for identification and quantification of antimicrobial peptides (AMPs) in biological samples. Amphipathic AMPs were extracted with various concentrations of ethanol, methanol, acetonitrile, formic acid, acetic acid or trichloroacetic acid in water. Yields were significantly greater for extraction with 66.7% ethanol than other extraction methods. Liquid chromatography was accomplished on a C18 column with a linear gradient of acetonitrile–formic acid, and mass spectrometry detection was performed in the positive electrospray multiple reaction monitoring mode by monitoring the transitions at m/z 385.2/239.2 and m/z 385.2/112.0 (AMP 1018), m/z 418.1/104.1 and m/z 418.1/175.1 (Methionine-1018). This method was shown to be reliable and efficient for the identification and quantification of scorpion AMPs Kn2-7 and its D-isomer dKn2-7 in human serum samples by monitoring the transitions at m/z 558.7/120.2 and m/z 558.7/129.1 (Kn2-7/dKn2-7).

Fungal infections are becoming a serious problem due to their high morbidity and mortality combined with the rise in drug resistance and dearth of new antimycotic drugs. The scorpion venom-derived peptide BmKn2, and its synthetic analogue Kn2–7, were previously observed to have antibacterial activity. These peptides and their d-amino acid analogues (dBmKn2 and dKn2–7) were tested for antifungal activity against drug resistant and clinical isolates of Candida albicans. In planktonic susceptibility studies, dKn2–7 had greater activity than the other three peptides against 6 out of 7 fungal strains, with no apparent correlation between drug resistance and minimum fungicidal concentrations (MFCs). Time kill experiments demonstrated that the fungicidal activity of dKn2–7 began within the first hour and killing rates were dose dependent at ≥ 1 × MFC. Against biofilms, the d-analogues were the most effective, while the l-analogues had low efficacy in most strains even at 10 times the planktonic MFC. Stability testing suggests that this increased efficacy of the d-analogues may be due to increased resistance to protease degradation compared to the l-analogues. Peptides were also assessed for mammalian cell toxicity. BmKn2 and dBmKn2 induced significant hemolysis at levels similar to their MFCs, whereas Kn2–7 and dKn2–7 caused hemolysis at 4–16 times their MFCs. The 50% inhibitory concentration (IC50) for dKn2–7 against murine fibroblasts was greater than or equal to the planktonic MFCs and biofilm IC50s for dKn2–7 in all C. albicans strains tested. These results support the potential for dKn2–7 to be further investigated as a novel antifungal therapeutic.