First results from a liquid-state shuttle dynamic nuclear polarization (DNP) spectrometer are presented. The device polarizes a water sample at 9.7 GHz and 0.34 T in a commercial Bruker electron paramagnetic resonance (EPR) spectrometer and then transfers the sample via a homebuilt pneumatic shuttle device into the 600 MHz and 14.09 T nuclear magnetic resonance (NMR) spectrometer with a conventional NMR probe for detection. The shuttle transfer time is approximately 115 ms. Initial experiments measure the postshuttle proton magnetization compared with the Boltzmann magnetization at 14.09 T. The DNP enhancement factor at 0.34 T is reported for the nitroxide polarizer TEMPOL in water solution. Reduction of the magnetization during shuttling because of relaxation is quantified. Optimization of this apparatus is expected to bring the NMR enhancement factor for protons close to the theoretical enhancement maximum of #7.92.
Venomous organisms have evolved a variety of structurally diverse peptide neurotoxins that target ion channels. Despite the lack of any obvious structural homology, unrelated toxins that interact with voltage-activated K(+) channels share a dyad motif composed of a lysine and a hydrophobic amino acid residue, usually a phenylalanine or a tyrosine. kappaM-Conotoxin RIIIK (kappaM-RIIIK), recently characterized from the cone snail Conus radiatus, blocks Shaker and TSha1 K(+) channels. The functional and structural study presented here reveals that kappaM-conotoxin RIIIK blocks voltage-activated K(+) channels with a novel pharmacophore that does not comprise a dyad motif. Despite the quite different amino acid sequence and no overlap in the pharmacological activity, we found that the NMR solution structure of kappaM-RIIIK in the C-terminal half is highly similar to that of mu-conotoxin GIIIA, a specific blocker of the skeletal muscle Na(+) channel Na(v)1.4. Alanine substitutions of all non-cysteine residues indicated that four amino acids of kappaM-RIIIK (Leu1, Arg10, Lys18, and Arg19) are key determinants for interaction with K(+) channels. Following the hypothesis that Leu1, the major hydrophobic amino acid determinant for binding, serves as the hydrophobic partner of a dyad motif, we investigated the effect of several mutations of Leu1 on the biological function of kappaM-RIIIK. Surprisingly, both the structural and mutational analysis suggested that, uniquely among well-characterized K(+) channel-targeted toxins, kappaM-RIIIK blocks voltage-gated K(+) channels with a pharmacophore that is not organized around a lysine-hydrophobic amino acid dyad motif.
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