Type IV pili are polymeric fibers which protrude from the cell surface and play a critical role in adhesion and invasion by pathogenic bacteria. The secretion of pili across the periplasm and outer membrane is mediated by a specialized secretin protein, PilQ, but the way in which this large channel is formed is unknown. Using NMR, we derived the structures of the periplasmic domains from N. meningitidis PilQ: the N-terminus is shown to consist of two β-domains, which are unique to the type IV pilus-dependent secretins. The structure of the second β-domain revealed an eight-stranded β-sandwich structure which is a novel variant of the HSP20-like fold. The central part of PilQ consists of two α/β fold domains: the structure of the first of these is similar to domains from other secretins, but with an additional α-helix which links it to the second α/β domain. We also determined the structure of the entire PilQ dodecamer by cryoelectron microscopy: it forms a cage-like structure, enclosing a cavity which is approximately 55 Å in internal diameter at its largest extent. Specific regions were identified in the density map which corresponded to the individual PilQ domains: this allowed us to dock them into the cryoelectron microscopy density map, and hence reconstruct the entire PilQ assembly which spans the periplasm. We also show that the C-terminal domain from the lipoprotein PilP, which is essential for pilus assembly, binds specifically to the first α/β domain in PilQ and use NMR chemical shift mapping to generate a model for the PilP:PilQ complex. We conclude that passage of the pilus fiber requires disassembly of both the membrane-spanning and the β-domain regions in PilQ, and that PilP plays an important role in stabilising the PilQ assembly during secretion, through its anchorage in the inner membrane.

Despite osteoarthritis (OA) and rheumatoid arthritis (RA) being typically age-related, their underlying etiologies are markedly different. We used 1H nuclear magnetic resonance (NMR) spectroscopy to identify differences in metabolite profiles in low volumes of OA and RA synovial fluid (SF). SF was aspirated from knee joints of 10 OA and 14 RA patients. 100 μL SF was analyzed using a 700 MHz Avance IIIHD Bruker NMR spectrometer with a TCI cryoprobe. Spectra were analyzed by Chenomx, Bruker TopSpin and AMIX software. Statistical analysis was undertaken using Metaboanalyst. 50 metabolites were annotated, including amino acids, saccharides, nucleotides and soluble lipids. Discriminant analysis identified group separation between OA and RA cohorts, with 32 metabolites significantly different between OA and RA SF (false discovery rate (FDR) < 0.05). Metabolites of glycolysis and the tricarboxylic acid cycle were lower in RA compared to OA; these results concur with higher levels of inflammation, synovial proliferation and hypoxia found in RA compared to OA. Elevated taurine in OA may indicate increased subchondral bone sclerosis. We demonstrate that quantifiable differences in metabolite abundance can be measured in low volumes of SF by 1H NMR spectroscopy, which may be clinically useful to aid diagnosis and improve understanding of disease pathogenesis.

Noncovalent interactions, such as hydrogen bonding, electrostatic, p-p, CH-p, and hydrophobic forces, play an essential role in the action of natures catalysts, enzymes. In the last decade these interactions have been successfully exploited in organocatalysis with small organic molecules. [1] In contrast, such interactions have rarely been studied in the wellestablished area of organometallic catalysis, [2] where electronic interactions through covalent bonding and steric effects imposed by bound ligands dictate the activity and selectivity of a metal catalyst. An interesting question is: What happens when an organocatalyst meets an organometallic catalyst? This unification has already created an exciting new space for both fields: cooperative catalysis, where reactants are activated simultaneously by both types of catalyst, thereby enabling reactivity and selectivity patterns inaccessible within each field alone. [3] However, the mechanisms by which the two catalysts cooperatively effect the catalysis remain to be delineated. We recently found that combining an achiral iridium catalyst with a chiral phosphoric acid allows for highly enantioselective hydrogenation of imines (Scheme 1). [4] To gain insight into the mechanism of this metal-organo cooperative catalysis, we studied the catalytic system with a range of techniques, including high pressure 2D-NMR spectroscopy, diffusion measurements, and NOEconstrained computation. Herein we report our findings.To evaluate the mechanism, a simplified achiral complex C was used, which leads to [C + ][A À ] upon mixing, in situ or ex situ, with the chiral phosphoric acid HA through protonation at the amido nitrogen (Scheme 1). In the asymmetric hydrogenation of the model ketimine 1 a, [C + ][A À ] afforded 95 % ee and full conversion. On the basis of related studies, [5] the hydrogenation can be broadly explained by the catalytic cycle shown in Scheme 1, that is, [C + ][A À ] activates H 2 to give the hydride D and protonated 1 a, which forms an ion pair with the phosphate affording [1 a + ][A À ]; [6,7] hydride transfer furnishes the amine product 2 a while regenerating [C + ][A À ]. Questions pertinent to possible iridium-phosphate cooperation then arise: 1) How does the chiral phosphoric acid induce asymmetry in the hydrogenation? and 2) Does the enantioselectivity result from D being formed enantioselectively from [C + ][A À ], from the phosphate salt [1 a + ][A À ], or from interactions involving all three components?We looked first at how the formation of hydride D and its transfer into the substrate are influenced by the chiral acid HA. The studies were carried out in CH 2 Cl 2 or CD 2 Cl 2 owing to the low solubility of the various metal complexes in toluene. The catalytic hydrogenation is feasible in both solvents, giving a 95 % ee in toluene and 85 % ee in CH 2 Cl 2 in the case of hydrogenation of 1 a with C and HA under the conditions given in Scheme 1. The solution NMR studies show that the ionic complex [C + ][A À ] is formed instantly on protonation of C (...

Osteoarthritis (OA), osteochondrosis (OC), and synovial sepsis in horses cause loss of function and pain. Reliable biomarkers are required to achieve accurate and rapid diagnosis, with synovial fluid (SF) holding a unique source of biochemical information. Nuclear magnetic resonance (NMR) spectroscopy allows global metabolite analysis of a small volume of SF, with minimal sample preprocessing using a noninvasive and nondestructive method. Equine SF metabolic profiles from both nonseptic joints (OA and OC) and septic joints were analyzed using 1D 1H NMR spectroscopy. Univariate and multivariate statistical analyses were used to identify differential metabolite abundance between groups. Metabolites were annotated via 1H NMR using 1D NMR identification software Chenomx, with identities confirmed using 1D 1H and 2D 1H 13C NMR. Multivariate analysis identified separation between septic and nonseptic groups. Acetate, alanine, citrate, creatine phosphate, creatinine, glucose, glutamate, glutamine, glycine, phenylalanine, pyruvate, and valine were higher in the nonseptic group, while glycylproline was higher in sepsis. Multivariate separation was primarily driven by glucose; however, partial-least-squares discriminant analysis plots with glucose excluded demonstrated the remaining metabolites were still able to discriminate the groups. This study demonstrates that a panel of synovial metabolites can distinguish between septic and nonseptic equine SF, with glucose the principal discriminator.

PEGylation of therapeutic proteins is commonly used to extend half-lives and to reduce immunogenicity. However, reports of antibodies toward PEGylated proteins and of poly(ethylene glycol) (PEG) accumulation suggest that efficacy and safety concerns may arise. To understand the relationship among the pharmacology, immunogenicity, and toxicology of PEGylated proteins, we require knowledge of the disposition and metabolic fate of both the drug and the polymer moieties. The analysis of PEG by standard spectrophotometric or mass spectrometric techniques is problematic. Consequently, we have examined and compared two independent analytical approaches, based on gel electrophoresis and nuclear magnetic resonance (NMR) spectroscopy, to determine the biological fate of a model PEGylated protein, 40KPEG-insulin, within a rat model. Both immunoblotting with an antibody to PEG and NMR analyses (LOD 0.5 μg/mL for both assays) indicated that the PEG moiety remained detectable for several weeks in both serum and urine following intravenous administration of 40KPEG-insulin (4 mg/kg). In contrast, Western blotting with anti-insulin IgG indicated that the terminal half-life of the insulin moiety was far shorter than that of the PEG, providing clear evidence of conjugate cleavage. The application of combined analytical techniques in this way thus allows simultaneous independent monitoring of both protein and polymer elements of a PEGylated molecule. These methodologies also provide direct evidence for cleavage and definition of the chemical species present in biological fluids which may have toxicological consequences due to unconjugated PEG accumulation or immunogenic recognition of the uncoupled protein.

The aggregation and fibril deposition of amyloid proteins have been implicated in a range of neurodegenerative and vascular diseases, and yet the underlying molecular mechanisms are poorly understood. Here, we use a combination of cell-based assays, biophysical analysis, and atomic force microscopy to investigate the potential involvement of oxidative stress in aortic medial amyloid (AMA) pathogenesis and deposition. We show that medin, the main constituent of AMA, can induce an environment rich in oxidative species, increasing superoxide and reducing bioavailable nitric oxide in human cells. We investigate the role that this oxidative environment may play in altering the aggregation process of medin and identify potential posttranslational modification sites where site-specific modification and interaction can be unambiguously demonstrated. In an oxidizing environment, medin is nitrated at tyrosine and tryptophan residues, with resultant effects on morphology that lead to longer fibrils with increased toxicity. This provides further motivation to investigate the role of oxidative stress in AMA pathogenicity.

MUPs (major urinary proteins) play an important role in chemical signalling in rodents and possibly other animals. In the house mouse (Mus musculus domesticus) MUPs in urine and other bodily fluids trigger a range of behavioural responses that are only partially understood. There are at least 21 Mup genes in the C57BL/6 mouse genome, all located on chromosome 4, encoding sequences of high similarity. Further analysis separates the MUPs into two groups, the 'central' near-identical MUPs with over 97% sequence identity and the 'peripheral' MUPs with a greater degree of heterogeneity and approximately 20-30% non-conserved amino acids. This review focuses on differences between the two MUP sub-groups and categorizes these changes in terms of molecular structure and pheromone binding. As small differences in amino acid sequence can result in marked changes in behavioural response to the signal, we explore the potential of single amino acid changes to affect chemical signalling and protein stabilization. Using analysis of existing molecular structures available in the PDB we compare the chemical and physical properties of the ligand cavities between the MUPs. Furthermore, we identify differences on the solvent exposed surfaces of the proteins, which are characteristic of protein-protein interaction sites. Correlations can be seen between molecular heterogeneity and the specialized roles attributed to some MUPs.

The genomes of rats and mice both contain a cluster of multiple genes that encode small (18-20 kDa) eight-stranded β-barrel lipocalins that are expressed in multiple secretory tissues, some of which enter urine via hepatic biosynthesis. These proteins have been given different names, but are mostly generically referred to as MUPs (major urinary proteins). The mouse MUP cluster is increasingly well understood, and, in particular, a number of roles for MUPs in chemical communication between conspecifics have been established. By contrast, the literature on the rat orthologues is much less well developed and is fragmented. In the present review, we summarize current knowledge on the MUPs from the Norway (or brown) rat, Rattus norvegicus.