Pamela J. Seaton

Unprecedented concentration dependent chemical shift variation in1H-NMR studies: A caveat in the investigations of molecular recognition and structure elucidation

Abstract Our studies show that changes in concentration can lead to significant changes in the1H chemical shifts of non-exchangeable hydrogens, and thus, in the NMR spectra. An important observation, as described for quinoline and other aromatic compounds, is that different protons shift to different extents in the same solvent as the concentration of the solute is varied, leading to overlap/coalescence or cross over of proton signals. Chemical shift variations can be so pronounced that they lead to misleading 1D and 2D spectral signatures. Such concentration dependent spectral pattern changes have far reaching implications in the use of1H-NMR spectroscopy for structure elucidation, spectral characterization, determination of purity and the study of molecular recognition. Changes in concentration shift different protons to different extents, leading to coalescence or crossover of1H-NMR signals. Such spectral changes have far reaching implications for the use of1H-NMR spectroscopy for structure elucidation and study of molecular recognition. Download : Download full-size image

Lysylated phospholipids stabilize models of bacterial lipid bilayers and protect against antimicrobial peptides.

Aminoacylated phosphatidylglycerols are common lipids in bacterial cytoplasmic membranes. Their presence in Staphylococcus aureus has been linked to increased resistance to a number of antibacterial agents, including antimicrobial peptides. Most commonly, the phosphatidylglycerol headgroup is esterified to lysine, which converts anionic phosphatidylglycerol into a cationic lipid with a considerably increased headgroup size. In the present work, we investigated the interactions of two well-studied antimicrobial peptides, cecropin A and mastoparan X, with lipid vesicles composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), containing varying fractions of an aminoacylated phosphatidylethanolamine, a stable analog of the corresponding phosphatidylglycerol-derivative. To differentiate between the effects of headgroup size and charge on peptide-lipid interactions, we synthesized two different derivatives. In one, the headgroup was modified by the addition of lysine, and in the other, by glutamine. The modification by glutamine results in a phospholipid with a headgroup size comparable to that of the lysylated version. However, whereas lysyl-phosphatidylethanolamine (Lys-PE) is cationic, glutaminyl-phosphatidylethanolamine (Gln-PE) is zwitterionic. We found that binding of mastoparan X and cecropin A was not significantly altered if the content of aminoacylated phosphatidylethanolamines did not exceed 20mol.%, which is the concentration found in bacterial membranes. However, a lysyl-phosphatidylethanolamine content of 20mol% significantly inhibits dye release from lipid vesicles, to a degree that depends on the peptide. In the case of mastoparan X, dye release is essentially abolished at 20mol.% lysyl-phosphatidylethanolamine, whereas cecropin A is less sensitive to the presence of lysyl-phosphatidylethanolamine. These observations are understood through the complex interplay between peptide binding and membrane stabilization as a function of the aminoacylated lipid content. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

A fluorogenic peptide containing the processing site of human SARS corona virus S-protein: kinetic evaluation and NMR structure elucidation.

Human severe acute respiratory syndrome coronavirus (hSARS‐CoV) is the causative agent for SARS infection. Its surface glycoprotein (spike protein) is considered to be one of the prime targets for SARS therapeutics and intervention because its proteolytic maturation by a host protease is crucial for host–virus fusion. Using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS‐CoV spike protein (Abz‐755Glu‐Gln‐Asp‐Arg‐Asn‐Thr‐Arg‐Glu‐Val‐Phe‐Ala‐Gln766‐Tyx‐NH2) and in vitro studies, we show that besides furin, other PCs, like PC5 and PC7, might also be involved in this cleavage event. Through kinetic measurements with recombinant PCs, we observed that the peptide was cleaved efficiently by both furin and PC5, but very poorly by PC7. The cleavage could be blocked by a PC‐inhibitor, α1‐PDX, in a dose‐dependent manner. Circular dichroism spectra indicated that this peptide possesses a high degree of sheet structure. Following cleavage by furin, the sheet content increased, possibly at the expense of turn and random structures. 1H NMR spectra from 2D COSY and ROESY experiments under physiological buffer and pH conditions indicated that this peptide possesses a structure with a turn at its C‐terminal segment, close to the cleavage site. The data suggest that the cleavable peptide bond is located within the most exposed domain; this is supported by the nearby turn structure. Several strong to weak NMR ROESY correlations were detected, and a 3D structure of the spike IQF peptide that contains the crucial cleavage site R761↓E has been proposed.

Replacement of Menhaden Fish Meal Protein by Solvent-Extracted Soybean Meal Protein in the Diet of Juvenile Black Sea Bass Supplemented with or without Squid Meal, Krill Meal, Methionine, and Lysine

Abstract Three experiments were conducted to determine the extent to which menhaden fish meal protein (FMP) can be replaced by solvent-extracted soybean meal protein (SBP) in the diet of juvenile black sea bass Centropristis striata. Diets were formulated replacing FMP by SBP at 0, 10, 20, 30, 40, 50, and 60% (experiment 1) and 0, 60, 70, 80, 90, and 100% (experiment 2), with supplementation with squid meal, krill meal, and attractants in both experiments. Experiment 3 was designed to replace FMP by SBP at 40, 50, 60, 70, and 80% without supplemental squid and krill meal and at 60% and 70% with supplemental methionine and lysine. Diets were fed twice daily to triplicate groups of fish (N = 15 per group) in 75-L tanks containing recirculating seawater. Fish were fed for 6, 10, and 8 weeks in experiments 1, 2, and 3, respectively. No significant differences in body weight gain, feed efficiency, and survival were observed among treatments in experiment 1. In experiment 2, no significant differences in percen...

Branched phospholipids render lipid vesicles more susceptible to membrane-active peptides

Iso- and anteiso-branched lipids are abundant in the cytoplasmic membranes of bacteria. Their function is assumed to be similar to that of unsaturated lipids in other organisms - to maintain the membrane in a fluid state. However, the presence of terminally branched membrane lipids is likely to impact other membrane properties as well. For instance, lipid acyl chain structure has been shown to influence the activity of antimicrobial peptides. Moreover, the development of resistance to antimicrobial agents in Staphylococcus aureus is accompanied by a shift in the fatty acid composition toward a higher fraction of anteiso-branched lipids. Little is known about how branched lipids and the location of the branch point affect the activity of membrane-active peptides. We hypothesized that bilayers containing lipids with low phase transition temperatures would tend to exclude peptides and be less susceptible to peptide-induced perturbation than those made from higher temperature melting lipids. To test this hypothesis, we synthesized a series of asymmetric phospholipids that only differ in the type of fatty acid esterified at the sn-2 position of the lipid glycerol backbone. We tested the influence of acyl chain structure on peptide activity by measuring the kinetics of release from dye-encapsulated lipid vesicles made from these synthetic lipids. The results were compared to those obtained using vesicles made from S. aureus and Staphylococcus sciuri membrane lipid extracts. Anteiso-branched phospholipids, which melt at very low temperatures, produced lipid vesicles that were only slightly less susceptible to peptide-induced dye release than those made from the iso-branched isomer. However, liposomes made from bacterial phospholipid extracts were generally much more resistant to peptide-induced perturbation than those made from any of the synthetic lipids. The results suggest that the increase in the fraction of anteiso-branched fatty acids in antibiotic-resistant strains of S. aureus is unlikely to be the sole factor responsible for the observed increased antibiotic resistance. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

N-(2-Aminobenzyl)-N,N-bis(quinolin- 2-ylmethyl)amine

The title new diquinaldine derivative, C27H24N4, forms molecular assemblies organized by intermolecular quinoline pi-pi stacking [3.356 (3) and 3.440 (3) A] and both inter- and intramolecular N-H...N hydrogen bonds [3.039 (3)-3.104 (3) A and 129 (2)-172 (2) degrees]. The combination of such interactions provides readily definable contacts that propagate along each crystallographic axis.