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 (RID) exchange properties of transmembrane peptides wit h varying length and composition. Synthetic transmembrane peptides were use d with a general acetyl-GW(2)(LA)(n)LW(2)A-ethanolamine sequence. These pep tides 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 a nalyzed by means of ESI-MS. First, the influence of the length of the hydro phobic Leu.-Ala sequence on exchange behavior was investigated. It was show n that longer peptide analogs are more protected from H/D exchange than exp ected on the basis of their length with respect to bilayer thickness. This is explained by an increased protection from the bilayer environment, be. c ause of stretching of the lipid acyl chains and/or tilting of the longer pe ptides. Next, the role of the flanking tryptophan residues was investigated . The length of the transmembrane part that shows very slow H/D exchange wa s found to depend on the exact position of the tryptophans in the peptide s equence, suggesting that tryptophan acts as a strong determinant for positi oning of proteins at the membrane/water interface. Finally, the influence o f putative helix breakers was studied. It was shown that the presence of Pr o in the transmembrane segment results in much higher exchange rates as com pared with Gly or Leu, suggesting a destabilization of the alpha -helix. Ta ndem MS measurements suggested that the increased exchange takes place over the entire transmembrane segment. The results show that ESI-MS is a conven ient technique to gain detailed insight into properties of peptides in lipi d bilayers by monitoring H/D exchange kinetics.