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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 C α proton with a methyl group, reduces the antimicrobial activity of GccF 1000-fold (IC 50 1.5 μM cf . 1.5 nM). A comparison of the GccF α-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.

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.

Bovine β-lactoglobulin (β-Lg) self-assembles into long amyloid-like fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). Heating β-Lg under fibril-forming conditions shows a lag phase before fibrils start forming. We have investigated the structural characteristics of β-Lg during the lag phase and the composition of β-Lg fibrils after their separation using ultracentrifugation. During the lag phase, the circular dichroism spectra of heated β-Lg showed rapid unfolding, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of samples showed increasing hydrolysis of β-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 β-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.

Campylobacter and Helicobacter species express a 6-amino-6-deoxyfutalosine N-ribosylhydrolase (HpMTAN) proposed to function in menaquinone synthesis. BuT-DADMe-ImmA is a 36 pM transition state analogue of HpMTAN, and the crystal structure of the enzyme-inhibitor complex reveals the mechanism of inhibition. BuT-DADMe-ImmA has a MIC(90) value of <8 ng/mL for Helicobacter pylori growth but does not cause growth arrest in other common clinical pathogens, thus demonstrating potential as an H. pylori-specific antibiotic.

The distribution and effect of applied strain on the collagen fibrils that make up leather may have an important bearing on the ultimate strength and other physical properties of the material. While sections of ovine and bovine leather were being subjected to tensile strain up to rupture, synchrotron-based small-angle X-ray scattering (SAXS) spectra were recorded edge-on to the leather at points from the corium to the grain. Measurements of both fibril orientation and collagen d spacing showed that, initially, the fibers reorient under strain, becoming more aligned. As the strain increases (5-10% strain), further fibril reorientation diminishes until, at 37% strain, the d spacing increases by up to 0.56%, indicating that significant tensile forces are being transmitted to individual fibrils. These changes, however, are not uniform through the cross-section of leather and differ between leathers of different strengths. The stresses are taken up more evenly through the leather cross-section in stronger leathers in comparison to weaker leathers, where stresses tended to be concentrated during strain. These observations contribute to our understanding of the internal strains and structural changes that take place in leather under stress.

Bacteriocins are bacterial peptides with specific activity against competing species. They hold great potential as natural preservatives and for their probiotic effects. We show here nuclear magnetic resonance-based evidence that glycocin F, a 43-amino acid bacteriocin from Lactobacillus plantarum, contains two β-linked N-acetylglucosamine moieties, attached via side chain linkages to a serine via oxygen, and to a cysteine via sulfur. The latter linkage is novel and has helped to establish a new type of post-translational modification, the S-linked sugar. The peptide conformation consists primarily of two α-helices held together by a pair of nested disulfide bonds. The serine-linked sugar is positioned on a short loop sequentially connecting the two helices, while the cysteine-linked sugar presents at the end of a long disordered C-terminal tail. The differing chemical and conformational stabilities of the two N-actetylglucosamine moieties provide clues about the possible mode of action of this bacteriostatic peptide.

SAXS has been applied to structural determination in leather. The SAXS beamline at the Australian Synchrotron provides 6 orders of magnitude dynamic range, enabling a rich source of structural information from scattering patterns of leather sections. SAXS patterns were recorded for q from 0.004 to 0.223 A(-1). Collagen d spacing varied across ovine leather sections from 63.8 nm in parts of the corium up to 64.6 nm in parts of the grain. The intensity of the collagen peak at q = 0.06 A(-1) varied by 1 order of magnitude across ovine leather sections with the high-intensity region in the corium and the low intensity in the grain. The degree of fiber orientation and the dispersion of the orientation has been quantified in leather. It is shown how the technique provides a wealth of useful information that may be used to characterize and compare leathers, skin, and connective tissue.

The effects of conventional lime sulfide depilation and enzymatic depilation on the enamel layer of pickled lamb pelts were examined using atomic force and optical microscopy, immunohistological, and proteomic techniques. Microscopy showed that the surface structure of enzymatically depilated material was visibly less organized than conventionally processed material, implying that the enzymes used for depilation were responsible for this difference. Proteomic analyses identified an absence of collagen VI in the enamel of skins that had been processed with enzymes, in contrast to the skins that had been processed using conventional methods, which was confirmed using immunolocalization studies. It is therefore possible that the destruction of collagen VI during enzymatic depilation may cause the changes to the enamel structure observed during enzyme processing and in turn affect the quality of the finished product.

Transition states can be predicted from an enzyme's affinity to related transition-state analogues. 5'-Methylthioadenosine nucleosidases (MTANs) are involved in bacterial quorum sensing pathways and thus are targets for antibacterial drug design. The transition-state characteristics of six MTANs are compared by analyzing dissociation constants (K(d)) with a small array of representative transition-state analogues. These inhibitors mimic early or late dissociative transition states with K(d) values in the picomolar range. Our results indicate that the K(d) ratio for mimics of early and late transition states are useful in distinguishing between these states. By this criterion, the transition states of Neisseria meningitides and Helicobacter pylori MTANs are early dissociative, whereas Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, and Klebsiella pneumoniae MTANs have late dissociative characters. This conclusion is confirmed independently by the characteristic [1'- (3)H] and [1'- (14)C] kinetic isotope effects (KIEs) of these enzymes. Large [1'- (3)H] and unity [1'- (14)C] KIEs are observed for late dissociative transition states, whereas early dissociative states showed close-to-unity [1'- (3)H] and significant [1'- (14)C] KIEs. K d values of various MTANs for individual transition-state analogues provide tentative information about transition-state structures due to varying catalytic efficiencies of enzymes. Comparing K d ratios for mimics of early and late transition states removes limitations inherent to the enzyme and provides a better predictive tool in discriminating between possible transition-state structures.

Oxazolidine E, an aldehydic cross-linking agent, is used to impart hydrothermal stability to collagen. The purpose of this study was to investigate the exact nature of oxazolidine E induced cross-links with collagen by using synthetic peptides having sequence homology with collagen type I. Tandem mass spectrometry revealed the formation of methylol and Schiff-base adducts upon reaction of oxazolidine E with the peptides. This was confirmed by allowing the reaction to proceed under reducing conditions using cyanoborohydride. Mass spectrometry (MS)-MS analysis clearly showed interaction of tryptophan and lysine residues with oxazolidine E and demonstrated that arginine could be cross-linked with glycine in the presence of oxazolidine E through the formation of a methylene bridge. Collagen fibrils regenerated from monomers in the presence and absence of oxazolidine E were studied using atomic force microscopy to investigate morphological alterations. Regenerated fibrils showing the typical 65 nm D-banding pattern were obtained from those formed both in the presence and absence of oxazolidine E, and there was no evidence of a change in the D-periodicity of these fibrils. This indicated that oxazolidine E does not hinder collagen molecules from correctly aligning to form the quarter-stagger structure.

3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and the four-carbon monosaccharide D-erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus is a member of the DAH7PS Ibeta subfamily, which also includes the KDO8PS enzymes. KDO8PS (3-deoxy-D-manno-octulosonate-8-phosphate synthase) catalyzes a closely related reaction of PEP with the five-carbon monosaccharide D-arabinose 5-phosphate (A5P). DAH7PS from P. furiosus requires a metal ion for activity and, unlike other characterized DAH7PS enzymes, is not inhibited by aromatic amino acids. Purified P. furiosus DAH7PS is able to utilize not only the four-carbon phosphorylated monosaccharides E4P and 2-deoxy-D-erythrose 4-phosphate but also the five-carbon phosphorylated monosaccharides A5P, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate with similar kcat but much increased KM values. DL-glyceraldehyde 3-phosphate and D-glucose 6-phosphate are not substrates. The structure of recombinant P. furiosus DAH7PS in complex with PEP was determined to 2.25 A resolution. The asymmetric unit consists of a dimer of (beta/alpha)8-barrel subunits. Analysis of the buried surfaces formed by dimerization and tetramerization, as observed in the crystal structure, provides insight into both the oligomeric status in solution and the substrate ambiguity of P. furiosus DAH7PS. P. furiosus DAH7PS is both the first archaeal and the first "naked" DAH7PS (without N-terminal extensions) to be fully characterized functionally and structurally. The broad substrate specificity of this DAH7PS, the lack of allosteric inhibition, and various structural features indicate that, of the enzymes characterized to date, P. furiosus DAH7PS may be the contemporary protein closest to the ancestral type I enzyme.

Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.