Solid-state NMR characterization of the putative membrane anchor of TWD1 from Arabidopsis thaliana
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- Scheidt, H.A., Vogel, A., Eckhoff, A. et al. Eur Biophys J (2007) 36: 393. doi:10.1007/s00249-006-0094-2
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Structure and membrane interaction of a 31 amino acid residue fragment of the membrane bound FKBP-like protein twisted dwarf 1 (TWD1) from Arabidopsis thaliana was investigated by solid-state NMR spectroscopy. The studied peptide TWD1(335–365) contained the putative membrane anchor of the protein (residues 339–357) that was previously predicted by sequence hydrophobicity analysis. The TWD1 peptide was synthesized by standard solid phase peptide synthesis and contained three uniformly 13C- and 15N-labelled residues (Phe 340, Val 350, Ala 364). The peptide was incorporated into either multilamellar vesicles or oriented planar membranes composed of an equimolar ternary phospholipid mixture (POPC, POPE, POPG), where the POPC was sn-1 chain-deuterated. 31P NMR spectra of the membrane in the absence and in the presence of the peptide showed axially symmetric powder patterns indicative of a lamellar bilayer phase. Further, the addition of peptide caused a decrease in the lipid hydrocarbon chain order as indicated by reduced quadrupolar splittings in the 2H NMR spectra of the POPC in the membrane. The conformation of TWD1(335–365) was investigated by 13C cross-polarization magic-angle spinning NMR spectroscopy. At a temperature of −30°C all peptide signals were resolved and could be fully assigned in two-dimensional proton-driven 13C spin diffusion and 13C single quantum/double quantum correlation experiments. The isotropic chemical shift values for Phe 340 and Val 350 exhibited the signature of a regular α-helix. Chemical shifts typical for a random coil conformation were observed for Ala 364 located close to the C-terminus of the peptide. Static 15N NMR spectra of TWD1(335–365) in mechanically aligned lipid bilayers demonstrated that the helical segment of TWD1(335–365) adopts an orientation perpendicular to the membrane normal. At 30°C, the peptide undergoes intermediate time scale motions.