G-PROTEIN-BOUND CONFORMATION OF MASTOPARAN-X - HETERONUCLEAR MULTIDIMENSIONAL TRANSFERRED NUCLEAR OVERHAUSER EFFECT ANALYSIS OF PEPTIDE UNIFORMLY ENRICHED WITH C-13 AND N-15

Mastoparans, a family of tetradecapeptides from wasp venom, have been used as convenient low molecular weight models of receptors coupled to GTP-binding regulatory proteins (G proteins) for the understanding of the interaction between G proteins and receptors. Sukumar and Higashi jima have analyzed the conformation of mastoparan-X (MP-X) bound to th e G protein alpha-subunit using proton two-dimensional transferred nuc lear Overhauser effect (TRNOE) spectroscopy [Sukumar, M., and Higashij ima, T. (1992) J. Biol. Chem., 267, 21421-21424]. The resultant struct ure, however, was not well-defined due to severe overlap of peptide pr oton resonances. To determine the G protein-bound conformation of MP-X in detail, we have analyzed this interaction by heteronuclear multidi mensional TRNOE experiments of MP-X uniformly enriched with N-15 and/o r C-13. By solving the overlap problem, we were able to determine the precise conformation of MP-X bound to Gil alpha: the peptide adopts an amphiphilic alpha-helix from Trp3 to C-terminal Leu14, and the atomic root-mean-square deviation (rmsd) values in this portion about the av eraged coordinates were 0.27 +/- 0.07 Angstrom for the backbone atoms (N, C-alpha, C') and 0.84 +/- 0.16 Angstrom for all heavy atoms. These values are much smaller than the corresponding rmsd values of the str uctures obtained from the proton 2D TRNOE spectrum alone: 1.70 +/- 0.4 1 Angstrom for the backbone atoms (N, C-alpha, C') and 2.84 +/- 0.51 A ngstrom for all heavy atoms. Our results indicate that the heteronucle ar multidimensional TRNOE experiments of peptides uniformly enriched w ith stable isotopes are a very powerful tool for analyzing the conform ation of short peptides bound to large proteins. We win also discuss t he structure-activity relationships of mastoparans in activating G pro teins on the basis of the precise structure of MP-X bound to Gil alpha .