Membrane Insertion of a Lipidated Ras Peptide Studied by FTIR, Solid-State NMR, and Neutron Diffraction Spectroscopy
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- 作者：Daniel Huster ; Alexander Vogel ; Catherine Katzka ; Holger A. Scheidt ; Hans Binder ; Silvia Dante ; Thomas Gutberlet ; Olaf Zschö ; rnig ; Herbert Waldmann ; and Klaus Arnold
- 刊名：Journal of the American Chemical Society
- 出版年：2003
- 出版时间：April 9, 2003
- 年：2003
- 卷：125
- 期：14
- 页码：4070 - 4079
- 全文大小：199K
- 年卷期：v.125,no.14(April 9, 2003)
- ISSN：1520-5126
文摘
Membrane binding of a doubly lipid modified heptapeptide from the C-terminus of the humanN-ras protein was studied by Fourier transform infrared, solid-state NMR, and neutron diffractionspectroscopy. The 16:0 peptide chains insert well into the 1,2-dimyristoyl-sn-glycero-3-phosphocholinephospholipid matrix. This is indicated by a common main phase transition temperature of 21.5 
C for boththe lipid and peptide chains as revealed by FTIR measurements. Further, 2H NMR reveals that peptideand lipid chains have approximately the same chain length in the liquid crystalline state. This is achievedby a much lower order parameter of the 16:0 peptide chains compared to the 14:0 phospholipid chains.Finally, proton/deuterium contrast variation of neutron diffraction experiments indicates that peptide chainsare localized in the membrane interior analogous to the phospholipid chains. In agreement with this modelof peptide chain insertion, the peptide part is localized at the lipid-water interface of the membrane. Thisis revealed by 1H nuclear Overhauser enhancement spectra recorded under magic angle spinning conditions.Quantitative cross-peak analysis allows the examination of the average location of the peptide backboneand side chains with respect to the membrane. While the backbone shows the strongest cross-relaxationrates with the phospholipid glycerol, the hydrophobic side chains of the peptide insert deeper into themembrane interior. This is supported by neutron diffraction experiments that reveal a peptide distributionin the lipid-water interface of the membrane. Concurring with these experimental findings, the amide protonsof the peptide show strong water exchange as seen in NMR and FTIR measurements. No indications fora hydrogen-bonded secondary structure of the peptide backbone are found. Therefore, membrane bindingof the C-terminus of the N-ras protein is mainly due to lipid chain insertion but also supported by interactionsbetween hydrophobic side chains and the lipid membrane. The peptide assumes a mobile and disorderedconformation in the membrane. Since the C-terminus of the soluble part of the ras protein is also disordered,we hypothesize that our model for membrane binding of the ras peptide realistically describes the membranebinding of the lipidated C-terminus of the active ras protein.
C for boththe lipid and peptide chains as revealed by FTIR measurements. Further, 2H NMR reveals that peptideand lipid chains have approximately the same chain length in the liquid crystalline state. This is achievedby a much lower order parameter of the 16:0 peptide chains compared to the 14:0 phospholipid chains.Finally, proton/deuterium contrast variation of neutron diffraction experiments indicates that peptide chainsare localized in the membrane interior analogous to the phospholipid chains. In agreement with this modelof peptide chain insertion, the peptide part is localized at the lipid-water interface of the membrane. Thisis revealed by 1H nuclear Overhauser enhancement spectra recorded under magic angle spinning conditions.Quantitative cross-peak analysis allows the examination of the average location of the peptide backboneand side chains with respect to the membrane. While the backbone shows the strongest cross-relaxationrates with the phospholipid glycerol, the hydrophobic side chains of the peptide insert deeper into themembrane interior. This is supported by neutron diffraction experiments that reveal a peptide distributionin the lipid-water interface of the membrane. Concurring with these experimental findings, the amide protonsof the peptide show strong water exchange as seen in NMR and FTIR measurements. No indications fora hydrogen-bonded secondary structure of the peptide backbone are found. Therefore, membrane bindingof the C-terminus of the N-ras protein is mainly due to lipid chain insertion but also supported by interactionsbetween hydrophobic side chains and the lipid membrane. The peptide assumes a mobile and disorderedconformation in the membrane. Since the C-terminus of the soluble part of the ras protein is also disordered,we hypothesize that our model for membrane binding of the ras peptide realistically describes the membranebinding of the lipidated C-terminus of the active ras protein.