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 amin o acids in this region, the effect of (-)charged phosphatidylserine at the concentration found naturally in the cytoplasmic leaflet (15 mol%) was cons idered. Each peptide contained 34 amino acids, which included the hydrophob ic 23 amino acid stretch thought to span the membrane and a ten amino acid segment beyond the 'cytoplasmic' surface. Non-perturbing deuterium probe nu clei were located within alanine side chains in intramembranous and extrame mbranous portions. H-2 NMR spectra were recorded at 35 degrees C and 65 deg rees C in fluid lipid bilayers consisting of (zwitterionic) 1-palmitoyl-2-o leoylphosphatidylcholin with and without 15 mol% (anionic) phosphatidylseri ne. 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 bot h types of membrane - as was the cc-helical intramembranous portion. Deuter ium 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 t he peptide backbone external to the membrane were qualitatively different f rom effects on the helical transmembrane domain - likely reflecting the dif ferent physical constraints on these peptide regions and the greater flexib ility of the extramembranous domain. Effects related to lipid charge could be detected in the spectrum of CD3 groups on the internally mobile side cha in of Val(650), six residues beyond the membrane surface. (C) 2000 Elsevier Science B.V. All rights reserved.