Measurement of interfluorine distances in solids

(19)F homonuclear dipolar recoupling methods were used to measure internuclear distances ranging from 5 to 12 A in fluorinated organic compounds in the solid state. Magic-angle-spinning-based high-resolution techniques were utilized. Trifluoromethyl and aromatic fluorine groups were separated by rigid aromatic spacers; these compounds were diluted into nonfluorinated host molecule matrices to give isolated homonuclear spin pairs with known internuclear distances. Radiofrequency-driven recoupling (RFDR) was used to elicit magnetization exchange between the spin pairs in 1D and 2D experiments. Simulation of the exchange was accomplished using a Monte Carlo-type algorithm to search the parameter space. These methods allow the determination of distances with an accuracy of 1 A at shorter distances and 2 A at longer distances, with the assumption of no prior knowledge of T(2)(ZQ).     

Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes

Converting lead compounds into drug candidates is a crucial step in drug development, requiring early assessment of potency, selectivity, and off-target effects. We have utilized activity-based chemical proteomics to determine the potency and selectivity of deubiquitylating enzyme (DUB) inhibitors in cell culture models. Importantly, we characterized the small molecule PR-619 as a broad-range DUB inhibitor, and P22077 as a USP7 inhibitor with potential for further development as a chemotherapeutic agent in cancer therapy. A striking accumulation of polyubiquitylated proteins was observed after both selective and general inhibition of cellular DUB activity without direct impairment of proteasomal proteolysis. The repertoire of ubiquitylated substrates was analyzed by tandem mass spectrometry, identifying distinct subsets for general or specific inhibition of DUBs. This enabled identification of previously unknown functional links between USP7 and enzymes involved in DNA repair.     

Bile UPLC-MS fingerprinting and bile acid fluxes during human liver transplantation

Bile flow restoration is a crucial step in the recovery process post transplantation of the liver. Here, metabolic trajectories based on changes in bile secretion - a known marker of functionality - have been utilised as an approach for discovering bile fluxes during transplantation. A total of ten liver transplants were monitored and from these 68 bile samples from both donors and recipients were collected and analysed using ultra-performance LC-MS in combination with multivariate statistical analysis. Based on the principal component scores constructed from the total bile fingerprint, differentiation of the bile acid concentrations before and after transplantation was detected. A trend was also observed, by constructing metabolic trajectories, whereby the post-transplant profiles approached the position of pre-transplant profiles within 30-60?min of the restoration of bile secretion function. The ten major conjugated bile acid salts were measured and a significant increase in concentrations of taurocholic acid and taurochenodeoxycholic acid were seen after transplantation. In addition, the ratios of secondary bile acids detected in gall bladder and hepatic bile were measured before and after transplantation. This study suggests that bile acid ratios in the donor liver at the pre-transplant and post-transplant stage may be important and that profiling of secreted bile after transplantation may aid clinical assessment and progress post-transplantation.     

Specific detection of Campylobacter jejuni using the bacteriophage NCTC 12673 receptor binding protein as a probe

Campylobacter jejuni is found in the intestines of poultry, cattle, swine, wild birds and pet animals and is the major cause of foodborne gastroenteritis in developed countries. We report the use of the receptor binding protein (RBP) of Campylobacter bacteriophage NCTC 12673 for the specific capture of Campylobacter jejuni bacteria using RBP-derivatized capturing surfaces. The Gp48 RBP was expressed as a glutathione S-transferase-Gp48 (GST-Gp48) fusion protein and immobilized onto surface plasmon resonance (SPR) surfaces using glutathione self-assembled monolayers (GSH SAM). Bovine serum albumin (BSA) was used to block any non-specific binding. Glutathione SAM leads to an oriented attachment of the protein, resulting in a two- to three-fold improvement of bacterial capture when compared to dithiobis(succinimidyl propionate) (DTSP) SAM-based unoriented attachment. The specificity of recognition was confirmed using Salmonella enterica subsp. enterica serovar Typhimurium as a negative control, which indeed showed negligible binding. The detection limit of the RBP-derivatized SPR surfaces was found to be 10(2) cfu/ml. Finally, GST-Gp48 was also immobilized onto magnetic beads that were successfully used to capture and pre-concentrate the host pathogen from suspension.     

Characterization of protein-ligand interactions by high-resolution solid-state NMR spectroscopy

A novel approach for detection of ligand binding to a protein in solid samples is described. Hydrated precipitates of the anti-apoptotic protein Bcl-xL show well-resolved (13)C-(13)C 2D solid-state NMR spectra that allow site-specific assignment of resonances for many residues in uniformly (13)C-enriched samples. Binding of a small peptide or drug-like organic molecule leads to changes in the chemical shift of resonances from multiple residues in the protein that can be monitored to characterize binding. Differential chemical shifts can be used to distinguish between direct protein-ligand contacts and small conformational changes of the protein induced by ligand binding. The agreement with prior solution-state NMR results indicates that the binding pocket in solid and liquid samples is similar for this protein. Advantages of different labeling schemes involving selective (13)C enrichment of methyl groups of Ala, Val, Leu, and Ile (Cdelta1) for characterizing protein-ligand interactions are also discussed. It is demonstrated that high-resolution solid-state NMR spectroscopy on uniformly or extensively (13)C-enriched samples has the potential to screen proteins of moderate size ( approximately 20 kDa) for ligand binding as hydrated solids. The results presented here suggest the possibility of using solid-state NMR to study ligand binding in proteins not amenable to solution NMR.