Dynamic Nuclear Polarization (DNP) experiments transfer polarization from electron spins to nuclear spins with microwave irradiation of the electron spins for enhanced sensitivity in nuclear magnetic resonance (NMR) spectroscopy. Design and testing of a spectrometer for magic angle spinning (MAS) DNP experiments at 263 GHz microwave frequency, 400 MHz 1H frequency is described. Microwaves are generated by a novel continuous-wave gyrotron, transmitted to the NMR probe via a transmission line, and irradiated on a 3.2 mm rotor for MAS DNP experiments. DNP signal enhancements of up to 80 have been measured at 95 K on urea and proline in water–glycerol with the biradical polarizing agent TOTAPOL. We characterize the experimental parameters affecting the DNP efficiency: the magnetic field dependence, temperature dependence and polarization build-up times, microwave power dependence, sample heating effects, and spinning frequency dependence of the DNP signal enhancement. Stable system operation, including DNP performance, is also demonstrated over a 36 h period.

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Neuropeptide Y (NPY), 1 a 36-residue peptide amide, is a member of the pancreatic polypeptide (PP) hormone family that also includes PP and peptide YY (PYY) (1). NPY is expressed in the central and peripheral nervous systems and is one of the most abundant neuropeptides in the brain. Several important physiological activities, such as induction of food intake, inhibition of anxiety, increase in memory retention, presynaptic inhibition of neurotransmitter release, vasoconstriction, and regulation of ethanol consumption, have been attributed to NPY (2, 3). Especially, the role of NPY in feeding is of major interest because NPY receptor antagonists are potential anti-obesity drug candidates. Many studies have established the strong central influence of NPY in feeding behavior; for example, injection of NPY into the hypothalamus increases food intake (4, 5), and high NPY levels are correlated with leptin deficiency (6), the hormone that is secreted by adipocytes and regulates body weight and energy balance (7,8). Furthermore, NPY knockout can reduce obesity in leptin-deficient mice (named ob/ob mice) (6).Five distinct Y receptor subtypes that bind NPY, PYY, and PP with different affinities have been identified, cloned, and characterized (9). They all belong to the superfamily of the G-protein- Because highly specific tools to investigate the Y 5 receptor activity are still missing, we have focused our work on the design of NPY receptor agonists with both high affinity and selectivity for the Y 5 subtype. It is well established that the C-terminal part of NPY represents the interaction site with the Y receptors and that amino acid exchange is poorly tolerated in the region 33-36 (49); therefore, we induced a conformational change within the peptide region that mediates receptor binding by introducing the ␤-turn-inducing dipeptide Ala-Aib (aminoisobutyric acid) (19) into positions 31-32 of NPY and some peptides that contain segments of NPY and PP (NPY/PP chimeras). The [Ala 31 ,Aib 32 ]-modified peptides showed high selectivity for the Y 5 receptor. Furthermore, in vitro and in vivo studies clearly proved their NPY receptor agonism as well as stimulation of food intake.

Protein amyloid fibrils can be functionalized by coating the core protofilament with high concentrations of proteins and enzymes. This can be done elegantly by appending a functional domain to an amyloidogenic protein monomer, then assembling the monomers into a fibril. To display an array of biologically functional porphyrins on the surface of protein fibrils, we have fused the sequence of the small, soluble cytochrome b562 to an SH3 dimer sequence that can form classical amyloid fibrils rapidly under well-defined conditions. The resulting fusion protein also forms amyloid fibrils and, in addition, binds metalloporphyrins, at half of the porphyrin binding sites as shown by UV-vis and NMR spectroscopies. Once metalloporphyrins are bound to the fibrils, the resulting holo-cytochrome domains are spectroscopically identical to the wild type cytochrome. The concentration of metalloporphyrins on a saturated fibril is estimated to be of the order of approximately 20 mM, suggesting that they could be interesting systems for applications in nanotechnology.

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