NMR Shows Why a Small Chemical Change Almost Abolishes the Antimicrobial Activity of Glycocin F.

Glycocin F (GccF), a ribosomally synthesized, post-translationally modified peptide secreted by Lactobacillus plantarum KW30, rapidly inhibits the growth of susceptible bacteria at nanomolar concentrations. Previous studies have highlighted structural features important for its activity and have shown the absolute requirement for the Ser18 O-linked GlcNAc on the eight-residue loop linking the two short helices of the (C-X6-C)2 structure. Here, we show that an ostensibly very small chemical modification to Ser18, the substitution of the Cα proton with a methyl group, reduces the antimicrobial activity of GccF 1000-fold (IC50 1.5 µM cf. 1.5 nM). A comparison of the GccFα-methylSer18 NMR structure (PDB 8DFZ) with that of the native protein (PDB 2KUY) showed a marked difference in the orientation and mobility of the loop, as well as a markedly different positioning of the GlcNAc, suggesting that loop conformation, dynamics, and glycan presentation play an important role in the interaction of GccF with as yet unknown but essential physiological target molecules.

School Nursing in a Pandemic: Striving for Excellence in Santa Fe Public Schools.

When the COVID-19 (coronavirus disease 2019) pandemic led to school closures around the nation in March 2020, the role of the school nurse changed significantly, and it has continued to evolve as districts grapple with how to safely meet students' academic needs while also protecting the health of their communities. Nurses working for Santa Fe Public Schools in New Mexico have taken their new roles seriously and have been working closely with their district leaders, the New Mexico Department of Health, School Health Advocates, and the Public Education Department to facilitate evidence-based policies and procedures. Activities have included cohorting, contact tracing, resource development, education (of staff and families), planning and implementation of safety procedures, coordination of surveillance testing, and staff screening, along with finding new, COVID safe ways to provide standard school nursing services, including immunization administration, hearing and vision screening, teaching, and promoting wellness and mental health.

Optimized Genetic Tools Allow the Biosynthesis of Glycocin F and Analogues Designed To Test the Roles of gcc Cluster Genes in Bacteriocin Production.

The emergence of multidrug-resistant pathogens has motivated natural product research to inform the development of new antimicrobial agents. Glycocin F (GccF) is a diglycosylated 43-amino-acid bacteriocin secreted by Lactobacillus plantarum KW30. It displays a moderate phylogenetic target range that includes vancomycin-resistant strains of Enterococcus species and appears to have a novel bacteriostatic mechanism, rapidly inhibiting the growth of the most susceptible bacterial strains at picomolar concentrations. Experimental verification of the predicted role(s) of gcc cluster genes in GccF biosynthesis has been hampered by the inability to produce soluble recombinant Gcc proteins. Here, we report the development of pRV610gcc, an easily modifiable 11.2-kbp plasmid that enables the production of GccF in L. plantarum NC8. gcc gene expression relies on native promoters in the cloned cluster, and NC8(pRV610gcc) produces mature GccF at levels similar to KW30. Key findings are that the glycosyltransferase glycosylates both serine and cysteine at either position in the sequence but glycosylation of the loop serine is both sequence and spatially specific, that glycosylation of the peptide scaffold is not required for export and subsequent disulfide bond formation, that neither of the putative thioredoxin proteins is essential for peptide maturation, and that removal of the entire putative response regulator GccE decreases GccF production less than removal of the LytTR domain alone. Using this system, we have verified the functions of most of the gcc genes and have advanced our understanding of the roles of GccF structure in its maturation and antibacterial activity.IMPORTANCE The entire 7-gene cluster for the diglycosylated bacteriocin glycocin F (GccF), including the natural promoters responsible for gcc gene expression, has been ligated into the Escherichia coli-lactic acid bacteria (LAB) shuttle vector pRV610 to produce the easily modifiable 11.2-kbp plasmid pRV610gcc for the efficient production of glycocin F analogues. In contrast to the refactoring approach, chemical synthesis, or chemoenzymatic synthesis, all of which have been successfully used to probe glycocin structure and function, this plasmid can also be used to probe in vivo the evolutionary constraints on glycocin scaffolds and their processing by the maturation pathway machinery, thus increasing understanding of the enzymes involved, the order in which they act, and how they are regulated.

Experimentally based structural model of Yih1 provides insight into its function in controlling the key translational regulator Gcn2.

Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1-Gcn2 interaction by binding to Gcn1, thus preventing Gcn2-mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules.

Qualitative and Quantitative Study of Glycosphingolipids in Human Milk and Bovine Milk Using High Performance Liquid Chromatography-Data-Dependent Acquisition-Mass Spectrometry.

Cerebrosides (Crb; including glucosylceramide and galactosylceramide) and lactosylceramide (LacCer) are structurally complex lipids found in many eukaryotic cell membranes, where they play important roles in cell growth, apoptosis, cell recognition and signaling. They are also found in mammalian milk as part of the milk fat globule membrane (MFGM), making milk an important dietary component for the rapidly growing infant. This study reports the development of a robust analytical method for the identification and characterization of 44 Crb and 23 LacCer molecular species in milk, using high performance liquid chromatography-tandem mass spectrometry in data-dependent acquisition mode. For the first time, it also compares the distributions of these species in human and bovine milks, a commercial MFGM-enriched dairy ingredient (MFGM Lipid 100) and commercial standards purified from bovine milk. A method for quantifying Crb and LacCer in milk using mass spectrometry in neutral loss scan mode was developed and validated for human milk, bovine milk and MFGM Lipid 100. Human milk was found to contain approximately 9.9-17.4 µg Crb/mL and 1.3-3.0 µg LacCer/mL, whereas bovine milk (pooled milk from a Friesian herd) contained 9.8-12.0 and 14.3-16.2 µg/mL of these lipids, respectively. The process used to produce MFGM Lipid 100 was shown to have enriched these components to 448 and 1036 µg/g, respectively. No significant changes in the concentrations of both Crb and LacCer were observed during lactation.

Bacteriocin ASM1 is an O/S-diglycosylated, plasmid-encoded homologue of glycocin F.

Here, we report on the biochemical characterization of a new glycosylated bacteriocin (glycocin), ASM1, produced by Lactobacillus plantarum A-1 and analysis of the A-1 bacteriocinogenic genes. ASM1 is 43 amino acids in length with Ser18-O- and Cys43-S-linked N-acetylglucosamine moieties that are essential for its inhibitory activity. Its only close homologue, glycocin F (GccF), has five amino acid substitutions all residing in the flexible C-terminal 'tail' and a lower IC50 (0.9 nm) compared to that of ASM1 (1.5 nm). Asm/gcc genes share the same organization (asmH← â†’asmABCDE→F), and the asm genes reside on an 11 905-bp plasmid dedicated to ASM1 production. The A-1 genome also harbors a gene encoding a 'rare' bactofencin-type bacteriocin. As more examples of prokaryote S-GlcNAcylation are discovered, the functions of this modification may be understood.

α-2'-Deoxyguanosine can switch DNA G-quadruplex topologies from antiparallel to parallel.

Here we demonstrate G-quadruplex formation by oligodeoxynucleotides containing α-2'-deoxyguanosine (α-dG) as a sole source of guanosines in G4T4, G4T4G4 and T(G3Tn)3G3T sequences with various numbers of natural ß-T in the loops (n = 1-4). Based on circular dichroism spectra we observed that all α-dG-containing DNAs formed G-quadruplexes with uniform arrangement of α-dG-tetrads, which implies formation of G-quadruplexes of parallel topology. In several cases, native DNA structures that usually adopt an antiparallel topology were converted to more thermally stable G-quadruplexes of parallel topology. Using 2D ROESY NMR spectra a new 'sequential walk' was established for α-dGs in a tetramolecular, parallel complex formed by the α-G4ß-T4 sequence. Analysis of ROEs in α-dGs indicates that guanines in [α-G4ß-T4]4 adopt anti-glycosidic conformations. These results demonstrate that α-dG can be used for an antiparallel-to-parallel switch of G-quadruplex DNAs producing complexes with higher thermal stability and uniform stacking of α-dG-tetrads.

Isolation and characterization of collagen type I crosslink from skin: high-resolution NMR reveals diastereomers of hydroxylysinonorleucine crosslink.

Skin is made up of mainly collagen type I and its structure is stabilised by the formation of covalent immature and mature crosslinks. In this study, collagen immature crosslink hydroxylysinonorleucine (HLNL) was isolated from bovine skin in high purity using two sequential purification steps. These consisted of preparative fibrous cellulose and size exclusion chromatography. The purified crosslink was then analysed using tandem mass spectrometry and high-resolution nuclear magnetic resonance (NMR) spectroscopy. The mass of singly and doubly charged ions of HLNL was 292.1865 and 146.5970 m/z and their optimised fragmentation energy was 17 keV and 5 keV, respectively. The 13C NMR of HLNL showed a doubled-up peak at 67.84 and 67.91 ppm which corroborated a diastereomeric form of collagen immature crosslink HLNL and both are chiroptically indistinguishable. The chemical structure was fully resolved using 1H, 13C and DEPT-135 high-resolution NMR spectroscopy and compared with other previous studies. We also obtained for the first time the 2D NMR spectra COSY and HSQC of HLNL. We therefore suggested that collagen organization into specific fibrils' orientation may be affected by the different configuration of these diastereomers of HLNL.

Molecular and structural insights into skin collagen reveals several factors that influence its architecture.

Although the biomechanical properties of skin and its molecular components have been extensively studied, little research has been devoted to understanding the links between them. Here, a comprehensive analysis of the molecular components of deer and cow skins was undertaken in order to understand the basis of their physical properties. These skins were chosen because they are known to be strong yet supple, exhibiting properties that have been exploited by man for centuries. Firstly, the tensile strength, tear strength and denaturation temperature of deer and cow skins were measured. Secondly, the organisation of the collagen fibrils and presence of glycosaminoglycans in each skin was investigated using polarising microscopy (PM), laser scanning confocal microscopy (LSCM), transmission electron microscopy (TEM), nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS). Finally, amino acid, crosslink and glycosaminoglycan analyses were carried out on both skins in the study. The results of the study showed that individual physical properties such as tensile strength of the skin are derived from different combinations of biomolecular components which are reflected in collagen architecture. The "wavy" organisation of collagen fibres in deer skin was associated with a small fibril diameter, uniform glycosaminoglycan distribution and higher proportion of trivalent crosslinks. In contrast, the collagen fibrils in cow skin were large, contained a diverse glycosaminoglycan distribution and a higher proportion of tetravalent crosslinks, resulting in straight fibres. This study showed for the first time that the relationship between the structure of collagen in skin and its biomechanical functions is complex, arising from different architectural and molecular features including organisation of collagen fibres, diameters of collagen fibrils, distribution and amount of glycosaminoglycans and types and concentrations of crosslinks.

Expression of Lactobacillus plantarum KW30 gcc genes correlates with the production of glycocin F in late log phase.

Antibacterial compounds known as bacteriocins are microbial inventions designed to reduce the competition for limited resources by inhibiting the growth of closely related bacteria. Glycocin F (GccF) is an unusually di-glycosylated bacteriocin produced in a lactic acid bacterium, Lactobacillus plantarum KW30 that has been shown to be resistant to extreme conditions. It is bacteriostatic rather than bactericidal, and all its post-translational modifications (a pair of nested disulfide bonds, and O-linked and S-linked N-acetylglucosamines) are required for full activity. Here, we examine a cluster of genes predicted to be responsible for GccF expression and maturation. The expression of eight genes, previously reported to make up the gcc operon, was profiled for their expression during cell culture. We found that all but one of the genes of the gcc cluster followed a pattern of expression that correlated with the stage of growth observed for the producer organism along with the increase in GccF secretion. We also found that most of the gcc genes are transcribed as a single unit. These data provide evidence that the gcc cluster genes gccABCDEF constitute a true operon for regulated GccF production, and explain the observed increase in GccF concentration that accompanies an increase in cell numbers.

Using Chemical Synthesis to Probe Structure-Activity Relationships of the Glycoactive Bacteriocin Glycocin F.

Glycocin F, a bacteriocin produced by Lactobacillus plantarum KW30, is glycosylated with two N-acetyl-d-glucosamine sugars, and has been shown to exhibit a rapid and reversible bacteriostasis on susceptible cells. The roles of certain structural features of glycocin F have not been studied to date. We report here the synthesis of various glycocin F analogues through solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL), allowing us to probe the roles of different structural features of this peptide. Our results indicate that the bacteriostatic activity of glycocin F is controlled by the glycosylated interhelical loop, while the glycosylated flexible tail appears to be involved in localizing the peptide to its cellular target.

Total chemical synthesis of glycocin F and analogues: S-glycosylation confers improved antimicrobial activity.

Glycocin F (GccF) is a unique diglycosylated bacteriocin peptide that possesses potent and reversible bacteriostatic activity against a range of Gram-positive bacteria. GccF is a rare example of a 'glycoactive' bacteriocin, with both the O-linked N-acetylglucosamine (GlcNAc) and the unusual S-linked GlcNAc moiety important for antibacterial activity. In this report, glycocin F was successfully prepared using a native chemical ligation strategy and folded into its native structure. The chemically synthesised glycocin appeared to be slightly more active than the recombinant material produced from Lactobacillus plantarum. A second-generation synthetic strategy was used to prepare 2 site selective 'glyco-mutants' containing either two S-linked or two O-linked GlcNAc moieties; these mutants were used to probe the contribution of each type of glycosidic linkage to bacteriostatic activity. Replacing the S-linked GlcNAc at residue 43 with an O-linked GlcNAc decreased the antibacterial activity, while replacing O-linked GlcNAc at position 18 with an S-linked GlcNAc increased the bioactivity suggesting that the S-glycosidic linkage may offer a biologically-inspired route towards more active bacteriocins.

Improved consistency in 2D gel electrophoresis: Sheep plasma as a test case.

Two-dimensional (2D) gel electrophoresis is a well-proven proteomic technique; however, sample-specific optimisation can often be necessary in order to get consistent quantitation. In particular, plasma samples are often smeared on 2D gels making spot matching difficult. A variety of different sample preparation and 2D methods were tested by using sheep plasma, and it was found that lowering sample pH prior to precipitation, using a long voltage gradient for isoelectric focusing and the inclusion of carrier ampholytes in the electrode wicks, improved both the quality and consistency of spot resolution. Analysis of the internal standards from two different DIGE experiments, one with conventional methodology and one with the improved method, showed that along with substantially improving the number of spots resolved, the average CV (coefficient of variation) of matched standards was lower with the new method. 428 matched spots were found using the improved method compared to 208 matched spots using conventional methodology. For the 174 spots that were matched between the two DIGE experiments, the average CV's of spot volumes were also significantly lower, at 0.20 for the new method compared to 0.24 for the conventional method (p < 0.001).

Liquid chromatography-electrospray ionization mass spectrometry for the simultaneous quantitation of collagen and elastin crosslinks.

We have developed a novel chromatographic analytical method for the simultaneous quantitation of collagen crosslinks. Seven non-derivatised crosslinks could be separated on a Cogent Diamond Hydride HPLC column using either isocratic or gradient conditions then detected by mass spectrometry. The total run time was less than 10min which is significantly shorter than that previously reported. This is the first method in which histidinohydroxylysinonorleucine (HHL) and histidinohydroxymero-desmosine (HHMD) were separated and identified by mass spectrometry without the need for pre- or post-column derivatization. The CVs of the retention times of all seven crosslinks were less than 1% and the limit of detection (LOD) and the limits of quantitation (LOQ) were 0.07-0.13pmol/µL and 0.20-0.38pmol/µL, respectively. This novel method was used for the routine analysis and quantitation of crosslinks in different animal skins in which potential new collagen crosslinks were identified that are as yet undocumented.

The glycocins: in a class of their own.

First reported in 2011, glycocins (glycosylated bacteriocins) are bacterial toxins that constitute a subset of ribosomally synthesised and post-translationally modified peptide (RiPP) natural products. Three NMR structures (glycocin F, ASM1 and sublancin 168), two with helix-loop-helix Cs α/α folds, are deposited in the PDB. Each structure contains a monosaccharide ß-S-linked to a cysteine side chain. Three more glycocins (thurandacin, and enterocins F4-9 and 96) have been biochemically characterised, and others predicted on the basis of bioinformatic analyses. Only glycocin F, ASM1 and enterocin F4-9 are unequivocally glycoactive. This review probes the structure-function relationships of four types of nested disulfide-bonded glycocins.

Synthesis of the antimicrobial S-linked glycopeptide, glycocin†F.

The first total synthesis of glycocin F, a uniquely diglycosylated antimicrobial peptide bearing a rare S-linked N-acetylglucosamine (GlcNAc) moiety in addition to an O-linked GlcNAc, has been accomplished using a native chemical ligation strategy. The synthetic and naturally occurring peptides were compared by HPLC, mass spectrometry, NMR and CD spectroscopy, and their stability towards chymotrypsin digestion and antimicrobial activity were measured. This is the first comprehensive structural and functional comparison of a naturally occurring glycocin with an active synthetic analogue.

Ultra-high resolution crystal structure of recombinant caprine ß-lactoglobulin.

ß-Lactoglobulin (ßlg) is the most abundant whey protein in the milks of ruminant animals. While bovine ßlg has been subjected to a vast array of studies, little is known about the caprine ortholog. We present an ultra-high resolution crystal structure of caprine ßlg complemented by analytical ultracentrifugation and small-angle X-ray scattering data. In both solution and crystalline states caprine ßlg is dimeric (K(D)<5 µM); however, our data suggest a flexible quaternary arrangement of subunits within the dimer. These structural findings will provide insight into relationships among structural, processing, nutritional and immunological characteristics that distinguish cow's and goat's milk.

Helicases, G4-DNAs, and drug design.

New helicase assays that recognise therapeutically important G4-DNA structures will lead to the discovery of novel molecular entities that bind not only to G4-tetrads, but also to grooves and loops of G4-DNA. Such assays can also provide inhibitors of G4-specific helicases that will shed light on the emerging involvement of helicases in cancer and other diseases linked to defective DNA repair pathways.

Β-lactoglobulin self-assembly: structural changes in early stages and disulfide bonding in fibrils.

Bovine ß-lactoglobulin (ß-Lg) self-assembles into long amyloid-like fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). Heating ß-Lg under fibril-forming conditions shows a lag phase before fibrils start forming. We have investigated the structural characteristics of ß-Lg during the lag phase and the composition of ß-Lg fibrils after their separation using ultracentrifugation. During the lag phase, the circular dichroism spectra of heated ß-Lg showed rapid unfolding, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of samples showed increasing hydrolysis of ß-Lg. The SDS-PAGE profiles of fibrils separated by ultra centrifugation showed that after six hours, the fibrils consisted of a few preferentially accumulated peptides. Two-dimensional SDS-PAGE under reducing and nonreducing conditions showed the presence of disulfide-bonded fragments in the fibrils. The sequences in these peptide bands were characterized by in-gel digestion electrospray ionization (ESI)-MS/MS. The composition of solubilized fibrils was also characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS/MS. Both MS analyses showed that peptides in fibrils were primarily from the N-terminal region, although there was some evidence of peptides from the C-terminal part of the molecule present in the higher molecular weight gel bands. We suggest that although the N-terminal region of ß-Lg is almost certainly involved in the formation of the fibrils, other peptide fragments linked through disulfide bonds may also be present in the fibrils during self-assembly.

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature.

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.