Solution structures of .alpha.-conotoxin G1 determined by two-dimensional NMR spectroscopy

Abstract

Two-dimensional NMR data have been used to generate solution structures of alpha-conotoxin G1, a potent peptide antagonist of the acetylcholine receptor. Structural information was obtained in the form of proton-proton internuclear distance constraints, and initial structures were produced with a distance geometry algorithm. Energetically more favorable structures were generated by using the distance geometry structures as input for a constrained energy minimization program. The results of both of these calculations indicate that the overall backbone conformation of the molecule is well-defined by the NMR data whereas the side-chain conformations are generally less well-defined. The main structural features derived from the NMR data were the presence of tight turns centered on residues Pro5 and Arg9. The solution structures are compared with previous proposed models of conotoxin G1, and the NMR data are interpreted in conjunction with chemical modification studies and structural properties of other antagonists of the acetylcholine receptor to gain insight into structure-activity relationships in these peptide toxins.

Two distinct structures of α‐conotoxin GI in aqueous solution

“…tions of 0.06Ϫ0.18 ppm from those reported by Pardi et al [9]. The absence of other significant (more than 0.05 ppm) devia-NMR structures of A-conotoxin GI in aqueous [9] and dimethylsulfoxide solution [10] have been previously studied.…”

“….. O 2.3 Å , N .. .O 3.3 Å ) and lower (NH .. .O 1.8 Å , N. .. O checked by matrix-assisted laser-desorption ionisation mass spectrometry. NMR spectra obtained under the conditions de-2.8 Å ) distance constraints to meet the hydrogen bond length and angle criteria; scribed in [9] demonstrated absence of impurities or disulfide isomers in the GI sample.…”

“…There are over-tected by butyloxycarbonyl groups, whereas side-chain funcall similarities between the X-ray structure and those derived at tional groups were blocked by benzyl-type groupings. The SHrelatively low resolution by NMR spectroscopy in water [9] and groups of cysteine residues were protected in the following man-( 2 H 6 )dimethylsulfoxide [10] solutions.ner: 4-methylbenzyl groups for Cys3 and Cys13, and It is well known that X-ray crystallography and NMR acetamidomethyl groups for Cys2 and Cys7. Fragment condenspectroscopy should provide complementary data on the spatial sation was performed with the aid of N-hydroxy-5-norboren-2,3-dicarboxyimide.…”

“…There are over-tected by butyloxycarbonyl groups, whereas side-chain funcall similarities between the X-ray structure and those derived at tional groups were blocked by benzyl-type groupings. The SHrelatively low resolution by NMR spectroscopy in water [9] and groups of cysteine residues were protected in the following man-( 2 H 6 )dimethylsulfoxide [10] solutions.…”

“…For this purpose we tested the con-D 2O. NMR data was processed with VNMR, Varian software, and sistency of experimental data set for each residue by comparing spin-spin coupling constant values with local distance conanalysis of the data was performed with the XEASY program [23]. Complete proton resonance assignment was done using a straints and calculating the local DIANA penalty function and R x -factor ; previously published assignment [9] with the XEASY program. Proton spin-spin coupling constants (12 H-NC A -H and 20 H-(b) generation of structure set by using standard approach (see above) on the basis of only static data (NOEs, torsion-angle C A C β -H) were measured in one-dimensional spectra.…”