Interfacial Positioning and Stability of Transmembrane Peptides in Lipid Bilayers Studied by Combining Hydrogen/Deuterium Exchange and Mass Spectrometry

Demmers, Jeroen; Duijn, Esther van; Haverkamp, Johan; Greathouse, Denise V.; Koeppe, Roger E.; Heck, Albert J. R.; Killian, J. Antoinette

doi:10.1074/jbc.m101401200

Cited by 77 publications

(97 citation statements)

References 27 publications

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“…The larger peptide has a positive hydrophobic mismatch and therefore already exposes a part of its hydrophobic R-helix in the interface (22). The barrier for removal is therefore more readily approached.…”

Section: Discussionmentioning

confidence: 99%

Strength of Integration of Transmembrane α-Helical Peptides in Lipid Bilayers As Determined by Atomic Force Spectroscopy

et al. 2004

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In this study we address the stability of integration of proteins in membranes. Using dynamic atomic force spectroscopy, we measured the strength of incorporation of peptides in lipid bilayers. The peptides model the transmembrane parts of R-helical proteins and were studied in both ordered peptiderich and unordered peptide-poor bilayers. Using gold-coated AFM tips and thiolated peptides, we were able to observe force events which are related to the removal of single peptide molecules out of the bilayer. The data demonstrate that the peptides are very stably integrated into the bilayer and that single barriers within the investigated region of loading rates resist their removal. The distance between the ground state and the barrier for peptide removal was found to be 0.75 ( 0.15 nm in different systems. This distance falls within the thickness of the interfacial layer of the bilayer. We conclude that the bilayer interface region plays an important role in stably anchoring transmembrane proteins into membranes.One out of three cellular proteins is a membrane protein because its sequence contains one or more stretches of hydrophobic amino acids that span the membrane as an R-helix. This motive allows for energetically favorable hydrogen bonding of the peptide backbone within the hydrophobic interior of the membrane. The hydrophobic helix in combination with flanking aromatic amino acids such as tryptophan ensures stable integration of intrinsic proteins into the lipid bilayer (1, 2). The bilayer is a complex and dynamic structure consisting of a hydrocarbon layer that is separated from the aqueous medium by a broad, water-rich interfacial region consisting of the headgroups, the glycerols, and ester bonds (1).Estimates on the membrane affinity of the hydrophobic helix are available (1). But precisely how stable are proteins integrated into membranes and what is the determinating factor for their stable integration? These are unanswered and fundamental questions in membrane biology that we address in this study.To get insight into these questions, a range of techniques such as optical tweezers (3) and biomembrane force probes (4) are available. Among these, atomic force microscopy (AFM) 1 is unique because it can combine spatial information with force measurements. Ideally, one would like to measure the strength of integration of a membrane protein in its natural surroundings. This has been done for bacteriorhodopsin and has resulted in insight into the unfolding pathway of this protein (5). However, interpretation of such studies in terms of contributions of specific parts of the protein and its relation to its surrounding is hampered by the complexity of such systems. Therefore, in this study we analyzed the strength of integration of transmembrane peptides consisting of a single R-helix that spans the bilayer. Such peptides can be designed to model the transmembrane parts of proteins and are commonly used in membrane research (6).The peptides used here are well characterized (6-8) and consist of a simp...

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Section: Discussionmentioning

confidence: 99%

Strength of Integration of Transmembrane α-Helical Peptides in Lipid Bilayers As Determined by Atomic Force Spectroscopy

et al. 2004

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“…Aromatics have both polar and hydrophobic characters and are known to favor the lipid-water interface location (33). Flanking aromatics confer resistance against displacement to experimental peptides embedded in bilayers (34,35). A survey of residue distribution shows interfacial preference for tryptophan, tyrosine, and histidine, although not for phenylalanine (36).…”

Section: Homologous Mutations In the Yeast Trpy1 And The Fly Trpc1 Havementioning

confidence: 99%

Yeast gain-of-function mutations reveal structure–function relationships conserved among different subfamilies of transient receptor potential channels

Su¹,

Zhou²,

Haynes³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…This would circumvent the need to quench the exchange reaction [6], transfer the sample into a solvent more suitable for measurements of hydrogen/deuterium populations [4] or use proteolytic digestion [23]. A new method introduced by Demmers and co-workers [10,11] meets these requirements by combining HDX of a proteoliposome suspension with nanoelectrospray ionization spectrometry (nano-ESI-MS). The work of this group has focused on the interactions between designed ␣-helical transmembrane peptides and large unilamellar vesicles (LUVs) formed by DMPC in water.…”

mentioning

confidence: 99%

Hydrogen/deuterium exchange of hydrophobic peptides in model membranes by electrospray ionization mass spectrometry

Hansen

Broadhurst

Skelton

et al. 2002

J. Am. Soc. Mass Spectrom.

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We demonstrate here that the hydrogen/deuterium solvent exchange (HDX) properties of the transmembrane fragment of the M2 protein of Influenza A (M2-TM) incorporated into lipid vesicles or detergent micelles can be studied with straightforward electrospray (ESI) and nanospray mass spectrometry (MS) configurations provided that key factors, including sample preparation techniques, are optimized. Small unilamellar vesicle preparations were obtained by solubilizing dimyristoyl phosphatidylcholine (DMPC) and the M2-TM peptide in aqueous solution with n-octyl-␤-D-glycopyranoside, followed by dialysis to remove the detergent. Electron microscopy experiments revealed that subsequent concentration by centrifugation introduced large multilamellar aggregates that were not compatible with ESI-MS. By contrast, a lyophilization-based concentration procedure, followed by thawing above the liquid crystal transition temperature of the lipid component, maintained the liposome size profile and yielded excellent ion fluxes in both ESI-MS and nano-ESI-MS. Using these methods the global HDX profile of M2-TM in aqueous DMPC vesicles was compared with that in methanol, demonstrating that several amide sites were protected from exchange by the lipid membrane. We also show that hydrophobic peptides can be detected by ESI-MS in the presence of a large molar excess of the detergent Triton X-100. The rate of HDX of M2-TM in Triton X-100 micelles was faster than that in DMPC vesicles but slower than when the peptide had been denatured in methanol. These results indicate that the accessibility of backbone amide sites to the solvent can be profoundly affected by membrane protein structure and dynamics, as well as the properties of model bilayer systems. (J Am Soc Mass Spectrom 2002, 13, 1376 -1387

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Interfacial Positioning and Stability of Transmembrane Peptides in Lipid Bilayers Studied by Combining Hydrogen/Deuterium Exchange and Mass Spectrometry

Cited by 77 publications

References 27 publications

Strength of Integration of Transmembrane α-Helical Peptides in Lipid Bilayers As Determined by Atomic Force Spectroscopy

Strength of Integration of Transmembrane α-Helical Peptides in Lipid Bilayers As Determined by Atomic Force Spectroscopy

Yeast gain-of-function mutations reveal structure–function relationships conserved among different subfamilies of transient receptor potential channels

Hydrogen/deuterium exchange of hydrophobic peptides in model membranes by electrospray ionization mass spectrometry

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