A Non-Covalent Dimer Formation of Quaternary Ammonium Cation with Unusual Charge Neutralization in Electrospray-Ionization Mass Spectrometry

Specific and nonspecific non-covalent molecular association of biomolecules is characteristic for electrospray-ionization mass spectrometry analysis of biomolecules. Understanding the interaction between two associated molecules is of significance not only from the biological point of view but also gas phase analysis by mass spectrometry. Here we reported a formation of non-covalent dimer of quaternary ammonium denatonium cation with +1 charge detected in the positive ion mode electrospray ionization mass spectrometry analysis of denatonium benzoate. Hydrogen deuterium exchange of amide and carbon-bonded hydrogens revealed that charge neutralization of one denatonium cation is the consequence of amide hydrogen dissociation. DFT (Density Functional Theory) calculations proved high thermodynamic stable of formed dimer stabilized by the short and strong N..H-N hydrogen bond. The signal intensity of the peak characterizing non-covalent dimer is low intensity and does not depend on the sample concentration. Additionally, dimer observation was found to be instrument-dependent. The current investigation is the first experimental and theoretical study on the quaternary ammonium ions dimer. Thus the present study has great significance for understanding the structures of the biomolecules as well as materials.

Dodecapeptide Cathelicidins of Cetartiodactyla: Structure, Mechanism of Antimicrobial Action, and Synergistic Interaction With Other Cathelicidins

In this study, dodecapeptide cathelicidins were shown to be widespread antimicrobial peptides among the Cetruminantia clade. In particular, we investigated the dodecapeptide from the domestic goat Capra hircus, designated as ChDode and its unique ortholog from the sperm whale Physeter catodon (PcDode). ChDode contains two cysteine residues, while PcDode consists of two dodecapeptide building blocks and contains four cysteine residues. The recombinant analogs of the peptides were obtained by heterologous expression in Escherichia coli cells. The structures of the peptides were studied by circular dichroism (CD), FTIR, and NMR spectroscopy. It was demonstrated that PcDode adopts a β-hairpin structure in water and resembles β-hairpin antimicrobial peptides, while ChDode forms a β-structural antiparallel covalent dimer, stabilized by two intermonomer disulfide bonds. Both peptides reveal a significant right-handed twist about 200 degrees per 8 residues. In DPC micelles ChDode forms flat β-structural tetramers by antiparallel non-covalent association of the dimers. The tetramers incorporate into the micelles in transmembrane orientation. Incorporation into the micelles and dimerization significantly diminished the amplitude of backbone motions of ChDode at the picosecond-nanosecond timescale. When interacting with negatively charged membranes containing phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), the ChDode peptide adopted similar oligomeric structure and was capable to form ion-conducting pores without membrane lysis. Despite modest antibacterial activity of ChDode, a considerable synergistic effect of this peptide in combination with another goat cathelicidin – the α-helical peptide ChMAP-28 was observed. This effect is based on an increase in permeability of bacterial membranes. In turn, this mechanism can lead to an increase in the efficiency of the combined action of the synergistic pair ChMAP-28 with the Pro-rich peptide mini-ChBac7.5Nα targeting the bacterial ribosome.

Characterization of the different oligomeric states of the DAN family antagonists SOSTDC1 and SOST

The DAN (differential screening-selected gene aberrative in neuroblastoma) family are a group of secreted extracellular proteins which typically bind to and antagonize BMP (bone morphogenetic protein) ligands. Previous studies have revealed discrepancies between the oligomerization state of certain DAN family members, with SOST (a poor antagonist of BMP signaling) forming a monomer while Grem1, Grem2, and NBL1 (more potent BMP antagonists) form non-disulfide linked dimers. The protein SOSTDC1 (Sclerostin domain containing protein 1) is sequentially similar to SOST, but has been shown to be a better BMP inhibitor. In order to determine the oligomerization state of SOSTDC1 and determine what effect dimerization might have on the mechanism of DAN family antagonism of BMP signaling, we isolated the SOSTDC1 protein and, using a battery of biophysical, biochemical, and structural techniques, showed that SOSTDC1 forms a highly stable non-covalent dimer. Additionally, this SOSTDC1 dimer was shown, using an in vitro cell based assay system, to be an inhibitor of multiple BMP signaling growth factors, including GDF5, while monomeric SOST was a very poor antagonist. These results demonstrate that SOSTDC1 is distinct from paralogue SOST in terms of both oligomerization and strength of BMP inhibition.

Membrane active cationic cholic acid-based molecular umbrellas

Herein, we report the synthesis of an umbrella thread and its covalent dimer and their transmembrane transport properties under physiological conditions.

Synthetic Covalently Linked Dimeric Form of H2 Relaxin Retains Native RXFP1 Activity and Has ImprovedIn VitroSerum Stability

Human (H2) relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles including regulation of connective tissue remodeling and systemic and renal vasodilation. These effects are mediated through interaction with its cognate G-protein-coupled receptor, RXFP1. H2 relaxin recently passed Phase III clinical trials for the treatment of congestive heart failure. However, itsin vivohalf-life is short due to its susceptibility to proteolytic degradation and renal clearance. To increase its residence time, a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chains, including a judiciously monoalkyne sited B-chain, followed by their combination through regioselective disulfide bond formation. Use of a bisazido PEG7linker and “click” chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered. The resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1 equally well. It had fewer propensities to activate RXFP2, the receptor for the related insulin-like peptide 3. In human serum, the dimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization may be a viable strategy for producing longer acting variants of H2 relaxin.