G Protein-Bound Conformation of Mastoparan-X: Heteronuclear Multidimensional Transferred Nuclear Overhauser Effect Analysis of Peptide Uniformly Enriched with 13C and 15N,

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Abstract
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 Higashijima have analyzed the conformation of mastoparan-X (MP-X) bound to the G protein α-subunit using proton two-dimensional transferred nuclear Overhauser effect (TRNOE) spectroscopy [Sukumar, M., and Higashijima, T. (1992) J. Biol. Chem., 267, 21421−21424]. The resultant structure, however, was not well-defined due to severe overlap of peptide proton resonances. To determine the G protein-bound conformation of MP-X in detail, we have analyzed this interaction by heteronuclear multidimensional TRNOE experiments of MP-X uniformly enriched with 15N and/or 13C. By solving the overlap problem, we were able to determine the precise conformation of MP-X bound to Gi1α: the peptide adopts an amphiphilic α-helix from Trp3 to C-terminal Leu14, and the atomic root-mean-square deviation (rmsd) values in this portion about the averaged coordinates were 0.27 ± 0.07 Å for the backbone atoms (N, Cα, C‘) and 0.84 ± 0.16 Å for all heavy atoms. These values are much smaller than the corresponding rmsd values of the structures obtained from the proton 2D TRNOE spectrum alone: 1.70 ± 0.41 Å for the backbone atoms (N, Cα, C‘) and 2.84 ± 0.51 Å for all heavy atoms. Our results indicate that the heteronuclear multidimensional TRNOE experiments of peptides uniformly enriched with stable isotopes are a very powerful tool for analyzing the conformation of short peptides bound to large proteins. We will also discuss the structure−activity relationships of mastoparans in activating G proteins on the basis of the precise structure of MP-X bound to Gi1α.