Structure–activity relationships of the antimicrobial peptide gramicidin S and its analogs: Aqueous solubility, self-association, conformation, antimicrobial activity and interaction with model lipid membranes

AbstractGS10 [cyclo-(VKLdYPVKLdYP)] is a synthetic analog of the naturally occurring antimicrobial peptide gramicidin (GS) in which the two positively charged ornithine (Orn) residues are replaced by two positively charged lysine (Lys) residues and the two less polar aromatic phenylalanine (Phe) residues are replaced by the more polar tyrosine (Tyr) residues. In this study, we examine the effects of these seemingly conservative modifications to the parent GS molecule on the physical properties of the peptide, and on its interactions with lipid bilayer model and biological membranes, by a variety of biophysical techniques. We show that although GS10 retains the largely β-sheet conformation characteristic of GS, it is less structured in both water and membrane-mimetic solvents. GS10 is also more water soluble and less hydrophobic than GS, as predicted, and also exhibits a reduced tendency for self-association in aqueous solution. Surprisingly, GS10 associates more strongly with zwitterionic and anionic phospholipid bilayer model membranes than does GS, despite its greater water solubility, and the presence of anionic phospholipids and cholesterol (Chol) modestly reduces the association of both GS10 and GS to these model membranes. The strong partitioning of both peptides into lipid bilayers is driven by a large favorable entropy change opposed by a much smaller unfavorable enthalpy change. However, GS10 is also less potent than GS at inducing inverted cubic phases in phospholipid bilayer model membranes and at inhibiting the growth of the cell wall-less bacterium Acholeplasma laidlawii B. These results are discussed in terms of the comparative antibiotic and hemolytic activities of these peptides

Development of paediatric quality of inpatient care indicators for low-income countries - A Delphi study

BACKGROUND: Indicators of quality of care for children in hospitals in low-income countries have been proposed, but information on their perceived validity and acceptability is lacking. METHODS: Potential indicators representing structural and process aspects of care for six common conditions were selected from existing, largely qualitative WHO assessment tools and guidelines. We employed the Delphi technique, which combines expert opinion and existing scientific information, to assess their perceived validity and acceptability. Panels of experts, one representing an international panel and one a national (Kenyan) panel, were asked to rate the indicators over 3 rounds and 2 rounds respectively according to a variety of attributes. RESULTS: Based on a pre-specified consensus criteria most of the indicators presented to the experts were accepted: 112/137(82%) and 94/133(71%) for the international and local panels respectively. For the other indicators there was no consensus; none were rejected. Most indicators were rated highly on link to outcomes, reliability, relevance, actionability and priority but rated more poorly on feasibility of data collection under routine conditions. There was moderate to substantial agreement between the two panels of experts. CONCLUSIONS: This Delphi study provided evidence for the perceived usefulness of most of a set of measures of quality of hospital care for children proposed for use in low-income countries. However, both international and local experts expressed concerns that data for many process-based indicators may not currently be available. The feasibility of widespread quality assessment and responsiveness of indicators to intervention should be examined as part of continued efforts to improve approaches to informative hospital quality assessment

Purification and Structural Characterization of Siderophore (Corynebactin) from Corynebacterium diphtheriae

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin

Separation of highly charged (+5 to +10) amphipathic α-helical peptide standards by cation-exchange and reversed-phase high-performance liquid chromatography

We are currently examining the potential of amphipathic cationic α-helical peptides as a new generation of peptide standards for both cation-exchange high-performance liquid chromatography and reversed-phase chromatography. Thus, amphipathic peptides are particularly suitable for high-performance liquid chromatography standards due to the preferred binding of the non-polar face to the hydrophobic stationary phase of reversed-phase packings or the preferred binding of the polar face to the charged/hydrophilic stationary phase of cation-exchange packings. The ability of different reversed-phase or cation-exchange matrices to separate mixtures of peptide standards with only subtle hydrophilicity/hydrophobicity variations in both the non-polar and polar face of the peptides can then be assessed. Currently, we have designed de novo a mixture of six 26-residue all D-conformation amphipathic cationic α-helical peptides with a single, positively charged lysine residue in the center of the non-polar face and an increasing number of lysine residues (4–9 residues) replacing neutral residues in the polar face, resulting in an overall net positive charge of +5 to +10. Thus, the non-polar, preferred reversed-phase chromatography binding face remains constant, with only the polar face varying in hydrophilicity/hydrophobicity. Interestingly, even with the non-polar face remaining constant, reversed-phase columns of varying functional group properties (e.g., C8, C18, phenyl, polar endcapped, polar embedded) and porosity (porous versus superficially porous) were able to separate the six peptides in aq. TFA/acetonitrile gradients, albeit with different selectivities. The value of the standards in cation-exchange chromatography was expressed by monitoring the requirement of acetonitrile (0–40% in the mobile phase) to overcome hydrophobic interactions of the peptides with the cation-exchange matrix matrix when eluting with sodium perchlorate gradients at pH 6.5. Interestingly, the resolution of the higher charged peptides (+8,+9,+10) was particularly sensitive to acetonitrile levels. Our results clearly demonstrate the excellent potential of these novel peptide standards to enable optimal column choice and mobile phase conditions for reversed-phase chromatography and cation-exchange chromatography for peptide separations

HPLC analysis and purification of peptides

High-performance liquid chromatography (HPLC) has proved extremely versatile over the past 25 yr for the isolation and punfication of peptides varying widely in their sources, quantity and complexity. This article covers the major modes of HPLC utilized for peptides (size-exclusion, ion-exchange, and reversed-phase), as well as demonstrating the potential of a novel mixed-mode hydrophilic interaction/cation-exchange approach developed in this laboratory. In addition to the value of these HPLC modes for peptide separations, the value of various HPLC techniques for structural characterization of peptides and proteins will be addressed, e.g., assessment of oligomerization state of peptideslproteins by sizeexclusion chromatography and monitoring the hydrophilicitykydrophobicity of amphipathic cr-helical peptides, a vital precursor Tor the development of novel antimicrobial peptides. The value of capillary electrophoresis for peptide separations is also demonstrated. Preparative reversed-phase chromatography purification protocols for sample loads of up to 200 mg on analytical columns and instrumentation are introduced for both peptides and recombinant proteins. Key Words: Peptides; proteins; size-exclusion chromatography (SEC); anion-exchange chromatography (AEX); cation-exchange chromatography (CEX); mixed-mode hydrophilic interaction chromatography (HIL1C)/cation-exchange chromatography (CEX); reversed-phase high-performance liquid chromatography (RP-HPLC); preparative RP-HPLC of peptides and proteins; amino acid side-chain hydrophilicitylhydrophobicity coefficients; amino acid U-helical propensity values; amino acid side-chain stability coefficient