Intermolecular interaction studies using small volumes.

We present the use of 1-mm room-temperature probe technology to perform intermolecular interaction studies using chemical shift perturbation methods and saturation transfer difference (STD) spectroscopy using small sample volumes. The use of a small sample volume (5-10 µl) allows for an alternative titration protocol where individual samples are prepared for each titration point, rather than the usual protocol used for a 5-mm probe setup where the ligand is added consecutively to the solution containing the protein or host of interest. This allows for considerable economy in the consumption and cost of the protein and ligand amounts required for interaction studies. For titration experiments, the use of the 1-mm setup consumes less than 10% of the ligand amount required using a 5-mm setup. This is especially significant when complex ligands that are only available in limited quantities, typically because they are obtained from natural sources or through elaborate synthesis efforts, need to be investigated. While the use of smaller volumes does increase the measuring time, we demonstrate that the use of commercial small volume probes allows the study of interactions that would otherwise be impossible to address by NMR.

Towards the conformational mimicry of the measles virus HNE loop: design, synthesis and biological evaluation of a cyclic bile acid-peptide conjugate.

The current article reports the design, synthesis and biochemical evaluation of a cyclic bile acid-peptide conjugate as a mimic of the loop-like structure of the measles virus haemagglutinin noose epitope (HNE). This macrocyclic structure was assembled by solid phase synthesis. Scaffold-peptide ring closure was achieved via the introduction of a succinate linker. After disulfide bridge formation with iodine, the desired 14 amino acid cyclic conjugate was obtained with overall yields between 15 and 35%. NMR analysis supports the presence of a helical conformation in the Q384-G388 pentapeptide portion, in agreement with the organisation of this chain in the native protein. The compound was found to have increased biostability compared to stabilised linear peptides, displayed good binding towards monoclonal antibodies known to bind to HNE and thus has potential in an alternative peptide-based measles vaccine.

Application of a combined weak cation-exchange/crown ether column: first demonstrations of a versatile tool for proteome subselection.

The present paper introduces the use of a weak cation-exchange/crown ether column in the proteomics field. The 18-crown-6 ether functionality is well-known to selectively complex ammonium and monoalkylammonium ions, which should make this column highly suitable to trap peptides with free alpha-NH(2) or free epsilon-NH(2) groups from lysine side chains. This unique selection mechanism was put to the test in an N-teromics setup which aims for the enrichment of deliberately acetylated protein N-terminal peptides from a serum digest. It was demonstrated that peptides with free alpha-NH(2) groups and peptides with alpha-amino-acetylated groups can be separated from each other using this weak cation-exchange/crown ether column. The peptides of interest, bearing no free primary amines, were found to be significantly enriched in the column's flow through. At the same time a favorable coenrichment of N-glycosylated peptides was observed. To obtain more insight in the contributions of the two distinct column functionalities, i.e., the weak cation exchanger and the crown ether, the experimental data were checked against a theoretical prediction of the outcome.

Rapid identification of common hexapyranose monosaccharide units by a simple TOCSY matching approach.

Solution NMR spectroscopy is a well established technique for non-destructive characterization of the structures and conformations of complex oligo- and polysaccharides. One of the key experiments involves the use of 2D TOCSY to collect the 1H spins into groups that can be associated with the individual saccharide units that are present in the molecule under study. It is well known that the magnetization transfer rate through the 1H spin system during the TOCSY spin lock period is sensitive to the intervening 3J(H,H) scalar couplings, and therefore also to the saccharide stereochemistry. Here, we have investigated the potential to extract information on the stereochemistry of hexapyranose monosaccharide units directly from TOCSY spectra. Through a systematic experimental investigation of the magnetization transfer initiated from the anomeric 1H resonance in D-glucose, D-galactose and D-mannose it is shown that a 100 ms spin lock time provides optimal spectroscopic discrimination between these three commonly occurring building blocks. A simple matching scheme is proposed as a new tool for rapid attribution of the TOCSY traces originating from the anomeric 1H resonances towards the underlying monosaccharide type. The scheme appears robust with regard to structural variations and fairly tolerant to incidental overlap. Its application provides useful guidance during the subsequent NMR assignment process, as demonstrated with the PS7F polysaccharide from Streptococcus pneumonia. In addition, we show that our scheme affords a clear-cut distinction between the alpha- and beta-epimers of D-mannose-type units, which can be difficult to discriminate by NMR analysis. Application to the N-glycan 100.2 demonstrates the potential and wide applicability of this new discrimination approach.