A 600 MHz 1H NMR study of toxin OSK1, blocker of small-conductance Ca2+-activated K+ channels, is presented. The unambiguous sequential assignment of all the protons of the toxin was obtained using TOCSY, DQF-COSY, and NOESY experiments at pH 3.0 (10, 30, and 45 degrees C) in aqueous solution. 3J(N alpha), 3J(alphabeta) vicinal spin coupling constants were determined in high-resolution spectra. The cross-peak volumes in NOESY spectra and the coupling constants were used to define the local structure of the protein by the program HABAS and to generate torsion angle and interproton distance constraints for the program DIANA. Hydrogen-deuterium exchange rates of amide protons showed possible locations of hydrogen bonds. The hydrogen bond acceptors and disulfide bridges between residues 8-28, 14-33, and 18-35 were determined when analyzing distance distribution in preliminary DIANA structures. All constraints were used to obtain a set of 30 structures by DIANA. The resulting rms deviations over 30 structures are 1.30 A for the heavy atoms and 0.42 A for the backbone heavy atoms. The structures were refined by constrained energy minimization using the SYBYL program. Their analysis indicated the existence of the alpha-helix (residues 10-21) slightly distorted at the Cys14 residue, two main strands of the antiparallel beta-sheet (24-29, 32-38), and the extended fragment (2-6). The motif is stabilized by the disulfide bridges in the way, common to all known scorpion toxins. Using the fine spatial toxin structure, alignment of the homologues, mutagenesis analysis, and comparison of scorpion toxin family functions, we delineate some differences significant for the toxin specificity.