title: Transmembrane region of the epidermal growth factor receptor: behavior and interactions via 2H NMR.
abstract: The first wide-line 2H NMR investigation of a receptor tyrosine kinase is reported. Selectively deuterated peptides from the membrane-associated portion of the human epidermal growth factor (EGF) receptor were synthesized for examination in lipid bilayers mimicking certain natural membrane features. The peptide sequence included the 23-amino acid hydrophobic stretch thought to span the membrane (Ile622-Met644 of the EGF receptor), plus the first 10 amino acids of the receptor's cytoplasmic domain (Arg645-Thr654). Dispersion of the peptide with lipid in the lipomimetic solvent, trifluoroethanol (TFE), was found to be a very useful initial step for sample preparation. TFE readily dissolved all components and was then easily removed in vacuo to yield thin films which could be subsequently hydrated to produce bilayers incorporating homogeneously dispersed peptide. Samples extensively studied consisted of 6 mol % peptide in multilamellar liposomes of 1-palmitoyl-2-oleoylphosphatidylcholine and similar liposomes containing cholesterol. 2H NMR spectra of the resulting unsonicated model membranes indicated the existence of peptide monomers undergoing rapid axially symmetric diffusion. It was possible to examine structural and behavioral effects of events often suggested as pivotal in signaling mechanisms and to consider by wide-line NMR for the first time the effect of cholesterol on hydrophobic peptides. When it was incorporated into bilayers by an alternative method involving dialysis of aqueous solutions prepared using a cationic detergent, spectra suggested that the peptide existed primarily as irreversibly aggregated oligomers which were relatively immobile on a time scale of 10(-3)-10(-4) s. For liposomes prepared by hydration of thin films, deuterated methyl groups on the peptide at locations corresponding to Ala623, Met644, and Val650 of the human EGF receptor were individually distinguishable. In highly fluid matrices, spectra suggested the presence of peptide monomers, diffusing symmetrically about axes perpendicular to the membrane. Studied as a function of temperature, 2H NMR spectra of such samples permitted independent consideration of membrane/peptide relationships at separate locations in the receptor tyrosine kinase. None of the locations probed demonstrated significant conformational sensitivity to temperature over a wide range. Effects seen at Ala623 and Met644, at opposite ends of the putative membrane-spanning domain, suggested slight increases in motional order with decreasing temperature. Addition of 33% cholesterol to the membrane caused little apparent conformational change at Val650 or Met644. However, in the presence of the sterol, Met644 and Ala623 exhibited nonaxially symmetric motion at low temperatures, perhaps as a result of peptide oligomerization. Moreover, the presence of cholesterol led to considerable change in spatial arrangement or order at Ala623. There was little evidence to support transmission of conformational changes along the peptide segment probed.
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, Canada.
authors.forename: A C
authors.initials: AC
authors.lastname: Rigby
authors.forename: K R
authors.initials: KR
authors.lastname: Barber
authors.forename: G S
authors.initials: GS
authors.lastname: Shaw
authors.forename: C W
authors.initials: CW
authors.lastname: Grant
mesh_terms: D000595:Amino Acid Sequence; D002784:Cholesterol / Q000494:pharmacology; D003903:Deuterium; D004134:Dimyristoylphosphatidylcholine; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines; D011487:Protein Conformation; D013696:Temperature; D014270:Trifluoroethanol / Q000494:pharmacology
chemical_list: D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D014270:Trifluoroethanol; D002784:Cholesterol; D003903:Deuterium; D066246:ErbB Receptors; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; D004134:Dimyristoylphosphatidylcholine