Sequence-related behaviour of transmembrane domains from class I receptor tyrosine kinases

Abstract2H NMR spectroscopy and freeze-fracture electron microscopy were used to compare the transmembrane domains of two Class I protein receptor tyrosine kinases (the EGF receptor and Neu/erbB-2) regarding overall behaviour in fluid lipid bilayer membranes. The 34-residue peptide, EGFRtm, was synthesised to contain the 23 amino acid hydrophobic stretch (Ile622 to Met644) thought to span the membrane of the human EGF receptor, plus the first 10 amino acids (Arg645 to Thr654) of the cytoplasmic domain. Deuterium probes replaced selected 1H nuclei at sites corresponding to Ala623, Met644, and Val650. The 38-residue peptide, Neutm, was synthesised having the 21 residue hydrophobic stretch (Ile660 to Ile680) calculated to span the membrane in rat Neu/erbB-2, plus residues Lys681 to Thr691 of the contiguous cytoplasmic domain. Deuterium probes replaced selected 1H nuclei at Ala661, Leu667, and Val676. A third peptide, Neutm*, was also prepared, corresponding to the transmembrane domain of a constitutively-activating Neu/erbB-2 transformant in which Val664 is replaced by Glu: it was deuterated in a manner identical to Neutm. Peptides were studied by 2H NMR spectroscopy at 1 mol% and 6 mol% in unsonicated fluid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and in POPC containing 33 mol% cholesterol, over the range 12° to 65°C. Overall motion was found to be different for each of the three peptides under a given set of conditions. EGFRtm spectra were characteristic of axially symmetric motion in membranes of POPC alone, and in POPC/cholesterol at 35°C and above. In contrast, spectra of the transmembrane peptides, Neutm and Neutm*, were characteristic of significantly axially asymmetric motion under all conditions studied (and regardless of sample preparation method). Addition of 33% cholesterol to membranes was accompanied by spectral changes consistent with increased formation of peptide dimers/oligomers in all cases. The transformant peptide, Neutm*, showed greater spectral evidence of immobilisation than did the wild type - probably reflecting a greater tendency to form large oligomers. Sequence-related details within the transmembrane domains of Class I receptor tyrosine kinases appear to exert important control over their associations within membranes. Freeze-fracture electron microscopy of the NMR samples demonstrated their liposomal nature. Peptide-related intramembranous particles (IMPs) were present which likely represent oligomers of the transmembrane peptide. IMP size and distribution were similar under a given set of conditions for all three peptides, suggesting that the differences seen by NMR spectroscopy reflect structures smaller than the 2 nm resolution limit of freeze-fracture EM and peptide relationships within its 20 nm accuracy of identifying lateral position