A transmembrane peptide from the human EGF receptor: behaviour of the cytoplasmic juxtamembrane domain in lipid bilayers

Solid state H-2 NMR spectroscopy was employed to study peptides related to the transmembrane domain of the human epidermal growth factor receptor, for insight into the interaction of its cytoplasmic juxtamembrane domain with the membrane surface. Since such receptors have clusters of (+)charged amino acids in this region, the effect of (-)charged phosphatidylserine at the concentration found naturally in the cytoplasmic leaflet (15 mol%) was considered. Each peptide contained 34 amino acids, which included the hydrophobic 23 amino acid stretch thought to span the membrane and a ten amino acid segment beyond the 'cytoplasmic' surface. Non-perturbing deuterium probe nuclei were located within alanine side chains in intramembranous and extramembranous portions. H-2 NMR spectra were recorded at 35 degrees C and 65 degrees C in fluid lipid bilayers consisting of (zwitterionic) 1-palmitoyl-2-oleoylphosphatidylcholin with and without 15 mol% (anionic) phosphatidylserine. The cationic extramembranous portion of the receptor backbone was found to be highly rotationally mobile on a time scale of 10(-4)-10(-5) s in both types of membrane - as was the cc-helical intramembranous portion. Deuterium nuclei in alanine side chains (-CD3) detected modest changes in peptide backbone orientation and/or dynamics related to the presence of 1-stearoyl-2-oleoylphosphatidylserine : in the case of the extramembranous portion of the peptide these seemed related to lipid charge. Temperature effects on the peptide backbone external to the membrane were qualitatively different from effects on the helical transmembrane domain - likely reflecting the different physical constraints on these peptide regions and the greater flexibility of the extramembranous domain. Effects related to lipid charge could be detected in the spectrum of CD3 groups on the internally mobile side chain of Val(650), six residues beyond the membrane surface. (C) 2000 Elsevier Science B.V. All rights reserved.