IONIZATION BEHAVIOR AND SOLUTION PROPERTIES OF THE POTASSIUM-CHANNEL BLOCKER SHK TOXIN

The effects of pH, temperature and polypeptide concentration on the so lution structure and side chain interactions of ShK toxin, a potassium -channel-blocking polypeptide from the sea anemone Stichodactyla la he lianthus, have been investigated by means of one-dimensional and two-d imensional H-1-NMR spectroscopy. Resonance assignments have been obtai ned for most protons in the molecule, and for the alpha and beta carbo n atoms. The lack of concentration dependence of the H-1 chemical shif ts and linewidths indicates that self-association is not significant a nd cannot account for the sheet-like structure near the N terminus. Th e structure is stable to high temperature, showing little change even at 353 K. This stability allowed backbone-amide temperature coefficien ts to be interpreted, and the correlation of these values with hydroge n bonds observed in the structures and with solvent exchange rates is discussed. pK(a) values have been measured for Asp5, His19 and Tyr23, and the contributions to these pK(a) values from other residues invest igated using the analogues R11Q (denoting substitution of Arg11 with G in), R11E, H19K, K22A, Y23A and K30A. These results show that Asp5 (pK (a) 2.8) makes an electrostatic interaction with Lys30, which may be p artially responsible for the importance of these side chains in the fo lding of synthetic toxin. The phenolic pK(a) of Tyr23 is reduced to 8. 7 in the native toxin, as a result of interactions with the positively charged side chains of Arg11 and to a lesser extent Lys22. Several hy drogen bonds between the Arg11 guanidino group and the Tyr23 phenolic group are found in the solution structures. As these three residues ar e implicated in the tight binding of ShK toxin to the T-lymphocyte vol tage-gated potassium channel Kv1.3, their close interactions should be taken into account in models of binding of this toxin to the pore and vestibule of this and other potassium channels.