Structure of Tightly Membrane-Bound Mastoparan-X, a G-Protein-Activating Peptide, Determined by Solid-State NMR

Abstract

The structure of mastoparan-X (MP-X), a G-protein activating peptide from wasp venom, in the state tightly bound to anionic phospholipid bilayers was determined by solid-state NMR spectroscopy. Carbon-13 and nitrogen-15 NMR signals of uniformly labeled MP-X were completely assigned by multidimensional intraresidue C-C, N-CalphaCbeta, and N-Calpha-C', and interresidue Calpha-CalphaCbeta, N-CalphaCbeta, and N-C'-Calpha correlation experiments. The backbone torsion angles were predicted from the chemical shifts of 13C', 13Calpha, 13Cbeta, and 15N signals with the aid of protein NMR database programs. In addition, two 13C-13C and three 13C-15N distances between backbone nuclei were precisely measured by rotational resonance and REDOR experiments, respectively. The backbone structure of MP-X was determined from the 26 dihedral angle restraints and five distances with an average root-mean-square deviation of 0.6 A. Peptide MP-X in the bilayer-bound state formed an amphiphilic alpha-helix for residues Trp3-Leu14 and adopted an extended conformation for Asn2. This membrane-bound conformation is discussed in relation to the peptide's activities to form pores in membranes and to activate G-proteins. This study demonstrates the power of multidimensional solid-state NMR of uniformly isotope-labeled molecules and distance measurements for determining the structures of peptides bound to lipid membranes.

Structure, Topology, and Tilt of Cell-Signaling Peptides Containing Nuclear Localization Sequences in Membrane Bilayers Determined by Solid-State NMR and Molecular Dynamics Simulation Studies

“…A typical 31 P 90°-pulse length of 3.5 μs was used. 31 P chemical shift spectra were obtained using a spin echo sequence (90°-τ-180°-τ-acquire with τ=9 μs), 65 kHz RF field for TPPM decoupling of protons, and a recycle delay of 3 seconds. A typical spectrum required the co-addition of about 100 scans for aligned samples and about 1,000 transients for MLVs.…”

“…Since the use of a high RF power in the PISEMA experiment could dehydrate the bilayer sample, cold air was circulated in the probe to reduce the effect of RF heat on the sample. In addition, the quality of aligned samples was examined using 1D 31 P experiments before and after PISEMA experiments. Data processing was accomplished using the Spinsight software (Varian) on a Sun Sparc workstation.…”

“…These results are also essential to interpret the NMR spectra of mechanically aligned lipid samples in terms of the peptide orientation. Therefore, 31 P chemical shift spectra of POPC and DMPC MLVs containing various concentrations (up to 5 mole %) of peptides were obtained at 30°C. Representative spectra are given in Figure 5.…”

Effects of different classes of attractants, cochliophilin A and <i>N</i>-(<i>E</i>)-feruloyl-4-<i>O</i>-methyldopamine, on the response of <i>Aphanomyces cochlioides</i> zoospores in their chemoattraction and activation of motility linked with intracellular cAMP

“…This clear difference in zoospore behavior and responses toward 1 and 2 in an aqueous solution of 10 also supports our tentative conclusion that the receptors of 1 and 2 are different, and these distinguishable sensing systems respectively play an important role in the events of host recognition and consequent infection of root tissues of the host plants. Islam et al 27) suggested that an important intracellular signaling cascade involved in the host-recognition system is likely to be mediated by cAMP, simply because treatment of the swimming zoospores with a G-protein activator, mastoparan, 28) led to their encystment followed by spore germination. Although it is simplistic thinking, we examined the effects of the adenylate cyclase activator forskolin (11) 18) and the adenylate cyclase …”

Characterization of the structure and dynamics of mastoparan‐X during folding in aqueous TFE by CD and NMR spectroscopy

“…10 It is one of a family of peptides that directly activate G proteins in a similar fashion to other G-protein coupled receptors. 11 Mastoparan-X is only partially folded in water, but it folds into an amphipathic helix when bound to membranes 12,13 or bound to the G protein a-subunit. 14 In both cases, the helix extends from residue 3 to residue 14 of the peptide.…”