Eurico J. Cabrita

Saturation transfer difference (STD) NMR technique and surface plasmon resonance (SPR) are used to study amino acid affinity supports–nucleotides interactions with l-histidine amino acid immobilized on a surface as model support. We have immobilized l-histidine ligand on a carboxymethyldextran- modified gold surface intended for surface plasmon resonance and we analyze the binding profiles of synthetic polynucleotides (1–6 base, sugar and backbone) by determining the equilibrium dissociation constant (KD). The SPR binding profile (square-shaped) is identical for all the complexes and the highest binding affinity can be found for polyA6 followed by polyG6 . As expected, the 5′ -mononucleotides have the lowest affinity. To further study the structural aspects of the interaction we investigate the polynucleotide binding preferences to l-histidine chromatography support by STD-NMR spectroscopy. These results revealed that an increase in the number of bases and backbone to 6 units leads to more contacts with the support, where the main driving force for the interaction with polynucleotides are through the base, except for polyC6 , which is mainly through sugar-phosphate backbone. Therefore, the combination of SPR measurements with STD-NMR technique allowed to establish fine details of the molecular recognition process involved in amino acid affinity supports–nucleotides complexes.

Saturation Transfer Difference-NMR (STD-NMR) spectroscopy has emerged as a powerful screening tool and a straightforward way to study the binding epitopes of active compounds in early stage lead discovery in pharmaceutical research. Here we report the application of STD NMR to characterize the binding of the anti-inflammatory drugs ibuprofen, diclofenac and ketorolac to COX-1 and COX-2. Using well-studied COX inhibitors and by comparing STD signals with crystallographic structures we show that there is a relation between the orientations of ibuprofen and diclofenac in the COX-2 active site and the relative STD responses detected in the NMR experiments. Based on this analysis we propose that ketorolac should bind to the COX-2 active site in similar orientation as that of diclofenac. We also show that the combination of STD NMR with competition experiments constitutes a valuable tool to address the recently proposed behavior of COX-2 as functional heterodimers and complement enzyme activity studies in the effort to rationalize COX inhibition mechanisms.

Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C4mim]Cl and [C2OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference – nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD.