Abstract
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.
Highlights
Nano-electrospray ionization mass spectrometry (ESIMS) was used to analyze hydrogen/deuterium (H/D) exchange properties of transmembrane peptides with varying length and composition
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
These populations are fast exchanging amide hydrogens located in the peptide termini that are exposed to the aqueous phase, intermediately exchanging hydrogens of the residues located in the bilayer/water interface, and slowly exchanging hydrogens located in the hydrophobic core of the lipid bilayer
Summary
Nano-electrospray ionization mass spectrometry (ESIMS) was used to analyze hydrogen/deuterium (H/D) exchange properties of transmembrane peptides with varying length and composition. A convenient way to gain insight into how the special characteristics of transmembrane segments and their interaction with lipids may influence the behavior of membrane proteins is by studying model systems of artificial transmembrane peptides with desired properties in well defined lipid bilayers. The results suggest that measurement of exchange properties of peptides by ESI-MS is a convenient method to investigate factors that determine interfacial positioning and/or stability of transmembrane protein segments. As models for protein transmembrane segments, we have used WALP peptides that already have been used successfully to investigate various aspects of peptide/lipid interactions [2,3,4] These peptides have a hydrophobic core of alternating Leu and Ala, which is flanked on both sides by Trp residues (see Table I), and they have been shown to form ␣-helical transmembrane helices [4]. Since in membrane proteins Trp residues are highly enriched near the membrane/water interface [5,6,7,8], they are thought to resemble a consensus sequence for
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