H(1)-NMR STUDY OF THE SOLUTION PROPERTIES AND SECONDARY STRUCTURE OF NEUROTOXIN-III FROM THE SEA-ANEMONE ANEMONIA-SULCATA

The solution properties, secondary structure and global fold of the 27 -residue polypeptide neurotoxin III (ATX III), from the sea anemone An emonia sulcata, have been investigated using high-resolution H-1-n.m.r . spectroscopy. Studies of the concentration dependence of the n.m.r. spectrum indicate that the molecule self-associates in the millimolar concentration range useable for n.m.r. analysis, the association being less pronounced at acidic pH values. The dependence on pH of associat ion implies that electrostatic interactions play a role in this proces s, while the significant concentration-dependent shifts of the aromati c resonances of Tyr-7 and Trp-13 indicate that hydrophobic interaction s also contribute. Individual pK(a) values have been determined for mo st ionizable groups in the molecule. Sequence-specific resonance assig nments were obtained for all protons using a range of two-dimensional homonuclear-correlated and nuclear-Overhauser-effect (nOe) spectra. Th e secondary structure of the polypeptide was identified from sequentia l (i, i + 1) and medium-range (i, i + 2/3/4) nOe connectivities, NH to C(alpha)H coupling constants, C(alpha)H chemical shifts, and the loca tion of slowly exchanging backbone-amide protons. ATX III contains no regular alpha-helix or beta-sheet, consisting instead of a network of reverse turns. nOe conectivities between half-cystine residues are con sistent with the disulphide pairings 3-17, 4-11 and 6-22. ATX III has a well-defined structure and appears to lack the disordered loop which , in the longer sea anemone toxins (46-49 residues ma be art of the re ceptor-binding surface.