Interfacial positioning and stability of transmembrane peptides in lipid bilayers studied by combining hydrogen/deuterium exchange and mass spectrometry.
Nano-electrospray ionization mass spectrometry (ESI-MS) was used to analyze hydrogen/deuterium (H/D) exchange properties of transmembrane peptides with varying length and composition. Synthetic transmembrane peptides were used with a general acetyl-GW(2)(LA)(n)LW(2)A-ethanolamine sequence. These peptides were incorporated in large unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The vesicles were diluted in buffered deuterium oxide, and the H/D exchange after different incubation times was directly analyzed by means of ESI-MS. First, the influence of the length of the hydrophobic Leu-Ala sequence on exchange behavior was investigated. It was shown that longer peptide analogs are more protected from H/D exchange than expected on the basis of their length with respect to bilayer thickness. This is explained by an increased protection from the bilayer environment, because of stretching of the lipid acyl chains and/or tilting of the longer peptides. Next, the role of the flanking tryptophan residues was investigated. The length of the transmembrane part that shows very slow H/D exchange was found to depend on the exact position of the tryptophans in the peptide sequence, suggesting that tryptophan acts as a strong determinant for positioning of proteins at the membrane/water interface. Finally, the influence of putative helix breakers was studied. It was shown that the presence of Pro in the transmembrane segment results in much higher exchange rates as compared with Gly or Leu, suggesting a destabilization of the alpha-helix. Tandem MS measurements suggested that the increased exchange takes place over the entire transmembrane segment. The results show that ESI-MS is a convenient technique to gain detailed insight into properties of peptides in lipid bilayers by monitoring H/D exchange kinetics.
Demmers
J A
JA
Department of Biochemistry of Membranes, Center for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
van Duijn
E
E
Haverkamp
J
J
Greathouse
D V
DV
Koeppe
R E
RE
Heck
A J
AJ
Killian
J A
JA
D000595:Amino Acid Sequence; D003903:Deuterium; D008051:Lipid Bilayers / Q000737:chemistry*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary
D008051:Lipid Bilayers; D008565:Membrane Proteins; D003903:Deuterium