With standard one- and two-dimensional proton NMR techniques, a common structural motif has been identified in water solutions of short peptide sequences derived from the envelope glycoprotein gp120 of HIV-1. Three peptides of lengths 12, 24, and 40 residues (termed RP342, RP142, and RP70, respectively) were synthesized, each containing a central amino acid sequence common to many HIV-1 isolates. In addition, RP70 contained a disulfide bond between cysteine residues close to the ends of the molecule, forming a loop that is thought to constitute an important structural and immunological component of the intact glycoprotein. Peptides RP70 and RP142 showed evidence for the presence of a significant population of conformations containing a beta-turn in the conserved sequence Gly-Pro-Gly-Arg. Strong nuclear Overhauser effect (NOE) connectivities were observed between the amide protons of the arginine and the adjacent glycine. A weak NOE connectivity was observed between the C alpha H of the proline residue and the NH of the Arg [a d alpha N(i,i + 2) NOE connectivity], confirming the presence of a conformational preference for a turn conformation in this sequence. The remainder of the peptide showed evidence of conformational averaging: no NMR evidence for a uniquely folded structure was obtained for any of the peptides in water solution. Circular dichroism (CD) spectra indicated that no ordered helix was present in water solutions of RP70, although a CD spectrum that indicated the presence of approximately 30% helix could be induced by the addition of trifluoroethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

The structure of the potassium channel blocker agitoxin 2 was solved by solution NMR methods. The structure consists of a triple-stranded antiparallel @-sheet and a single helix covering one face of the 0-sheet. The cysteine side chains connecting the &sheet and the helix form the core of the molecule. One edge of the 0-sheet and the adjacent face of the helix form the interface with the Shaker K+ channel. The fold of agitoxin is homologous to the previously determined folds of scorpion venom toxins. However, agitoxin 2 differs significantly from the other channel blockers in the specificity of its interactions. This study was thus focused on a precise characterization of the surface residues at the face of the protein interacting with the Shaker K+ channel. The rigid toxin molecule can be used to estimate dimensions of the potassium channel. Surface-exposed residues, ArgZ4, Lys", and Arg3' of the &sheet, have been identified from mutagenesis studies as functionally important for blocking the Shaker K + channel. The sequential and spatial locations of ArgZ4 and Arg3' are not conserved among the homologous toxins. Knowledge on the details of the channel-binding sites of agitoxin 2 formed a basis for site-directed mutagenesis studies of the toxin and the K+ channel sequences. Observed interactions between mutated toxin and channel are being used to elucidate the channel structure and mechanisms of channel-toxin interactions.Keywords: agitoxin; K+ channel; NMR; protein; scorpion toxin Scorpion venoms contain a large number of closely related peptide inhibitors of voltage-and Ca'+-dependent K+ channels. These peptides interact directly with the central catalytic region of K + channels-the ion conduction pore. They occlude the pore and prevent ion conduction by binding with one-to-one stoichiometry to the extracellular entryway of the channel (Miller et al., 1985;MacKinnon & Miller, 1988, 1989Miller, 1988;Giangiacomo et al., 1992). Knowledge of the precise structure and dimensions of scorpion toxins has been most valuable for probing the structure of K+ channels. The toxin molecules A.M. Krezel and C. Kasibhatla contributed equally to this work. Abbreviations: AgTxl, AgTx2, AgTx3, agitoxin variants 1, 2, and 3; ChTx, charybdotoxin; Lq2, Leiurus quinquestriatus variant 2 toxin; IbTx, iberiotoxin; KTx, kaliotoxin; MgTx, margatoxin; NxTx, noxiustoxin; Stv3, scorpion toxin variant-3; NOESY, NOE spectroscopy; HSQC, heteronuclear single-quantum coherence; TOCSY, total correlation spectroscopy; DQF-COSY, double quantum filtered correlated spectroscopy; 2D, two dimensional; 3D, three dimensional; HNHA and HNHB, ISN-separated quantitative J-correlation experiments; RMSD, RMS deviation. are used as calipers to measure the dimensions and geometry of the ion conduction pore regions of K+ channels.The known scorpion toxin K + channel inhibitors are 37-39 amino acids in length and contain six cysteine residues. They fall into three subclasses (Garcia et al., 1994). Within each subclass, toxins show greater than 70% amino a...

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A hybrid peptide of cecropin A and melittin was investigated by two-dimensional 'H-NMR at pH 5.8 in aqueous solution with 30% (by vol.) hexafluoroisopropanol. The peptide contains 26 amino acids, is a combination of the first 13 residues of each of the two parent peptides, CA(1-13)M(1-13) = CAM (1-26) 1061 and shown to have strong antibacterial activity but to be harmless towards erythrocytes. All resonances of the main chain and side chain /I-protons are assigned except for those of the N-terminal lysine. Several medium range NOE connectivities were observed showing two separated a-helices, involving residues 4-12 and 16-26. The J,,,-coupling constants in these sections support the conclusion. From the exchange rates of the NH protons it is concluded that the a-helix of residues 16 -26 is much more stable than the other helix. The circular dichroism data indicates about 30% less a-helix character than the NMR data. A reduced contribution to the ellipticity from the unstable helix is suggested.The chemical-shift differences between the two parts of the hybrid and the respective parent peptides are larger for the cecropin part than for the melittin part. For the latter, residues 17-26 of the hybrid are proposed to have a secondary structure very similar to that of residues 4-13 of melittin. The total results suggest that a hydrophobic a-helix capable of spanning half a lipid bilayer combined with an amphiphilic a-helix of two to three turns with a flexible hinge section in between are features of importance for the lytic antibacterial activity.

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