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• W2029159154 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-GW2(LA)nLW2A-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 α-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. 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-GW2(LA)nLW2A-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 α-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. nano-electrospray ionization mass spectrometry collision-induced dissociation, DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine hydrogen/deuterium large unilamellar vesicles prepared by extrusion tandem mass spectrometry mass-to-charge ratio The precise manner in which membrane proteins are embedded in a lipid bilayer is essential for their structure and function. Important structural and dynamic features, such as stability of the transmembrane segments or their precise positioning at the lipid/water interface, will be determined not only by intrinsic properties of the transmembrane segments, but also by their interaction with surrounding lipids. 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. Recently, we have described a new method using nano-ESI-MS1 (1Demmers J.A.A. Haverkamp J. Heck A.J.R. Koeppe R.E., II Killian J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3189-3194Crossref PubMed Scopus (103) Google Scholar) to study the properties of transmembrane protein segments in model systems by analyzing the kinetics of hydrogen/deuterium (H/D) exchange. The results showed that various populations of amide hydrogen atoms can be distinguished that are characteristic for different regions of the transmembrane segments. 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. 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. In the present study, we have investigated the influence of several of such factors. Hereby, special emphasis is given to the length of the α-helical hydrophobic core with respect to the bilayer thickness, the role of potential anchoring residues at the lipid/water interface, and the influence of α-helix breaking residues in the transmembrane segment. 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 (2de Planque M.R.R. Greathouse D.V. Koeppe R.E., II Schafer H. Marsh D. Killian J.A. Biochemistry. 1998; 37: 9333-9345Crossref PubMed Scopus (243) Google Scholar, 3de Planque M.R.R. Kruijtzer J.A.W. Liskamp R.M.J. Marsh D. Greathouse D.V. Koeppe R.E., II de Kruijff B. Killian J.A. J. Biol. Chem. 1999; 274: 20839-20846Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 4Killian J.A. Salemink I. de Planque M.R. Lindblom G. Koeppe R.E., II Greathouse D.V. Biochemistry. 1996; 35: 1037-1045Crossref PubMed Scopus (258) Google Scholar). 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 α-helical transmembrane helices (4Killian J.A. Salemink I. de Planque M.R. Lindblom G. Koeppe R.E., II Greathouse D.V. Biochemistry. 1996; 35: 1037-1045Crossref PubMed Scopus (258) Google Scholar). Since in membrane proteins Trp residues are highly enriched near the membrane/water interface (5Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A.L. Gulbis J.M. Cohen S.L. Chait B.T. Mackinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5649) Google Scholar, 6Kovacs F. Quine J. Cross T.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7910-7915Crossref PubMed Scopus (75) Google Scholar, 7Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1964) Google Scholar, 8Landolt Marticorena C. Williams K.A. Deber C.M. Reithmeier R.A. J. Mol. Biol. 1993; 229: 602-608Crossref PubMed Scopus (325) Google Scholar), they are thought to resemble a consensus sequence for transmembrane α-helical segments of intrinsic membrane proteins.Table IAmino acid sequences of the peptides used and their number of exchangeable hydrogensPeptideSequenceAverage Mass (Da)No. of labile H'sWALP16aAc, acetyl.Ac-GWWLALALALALAWWA-EtnbEtn, ethanolamine.1897.3122WALP19Ac-GWWLALALALALALALWWA-Etn2194.7125WALP21Ac-GWWLALALALALALALALWWA-Etn2379.9527WALP23Ac-GWWLALALALALALALALALWWA-Etn2563.1829W′ALP21dW′, Trp analog withN-methylindole side chain.Ac-GW′W′LALALALALALALALW′W′A-AmcAm, amide.2391.0023WALP23innerAc-GAWLALALALALALALALALWAA-Etn2332.9127WALP23outerAc-GWLALALALALALALALALALWA-Etn2374.9927WALP23ProAc-GWWLALALALAPALALALALWWA-Etn2547.1428WALP23GlyAc-GWWLALALALAGALALALALWWA-Etn2507.0729a Ac, acetyl.b Etn, ethanolamine.c Am, amide.d W′, Trp analog withN-methylindole side chain. Open table in a new tab The extent to which the hydrophobic length of transmembrane segments matches the hydrophobic bilayer thickness can significantly influence membrane protein structure and function (9Killian J.A. Biochim. Biophys. Acta. 1998; 1376: 401-415Crossref PubMed Scopus (510) Google Scholar). To investigate whether this is related to changes in membrane protein interfacial positioning and/or stability, we first analyzed the effects of increasing the hydrophobic length of WALP peptides on the H/D exchange kinetics of peptides with different lengths of the Leu-Ala core in bilayers of DMPC. This lipid was chosen because it forms well-defined bilayers with all peptides under experimental conditions (2de Planque M.R.R. Greathouse D.V. Koeppe R.E., II Schafer H. Marsh D. Killian J.A. Biochemistry. 1998; 37: 9333-9345Crossref PubMed Scopus (243) Google Scholar, 4Killian J.A. Salemink I. de Planque M.R. Lindblom G. Koeppe R.E., II Greathouse D.V. Biochemistry. 1996; 35: 1037-1045Crossref PubMed Scopus (258) Google Scholar, 10de Planque M.R.R. Goormaghtigh E. Greathouse D.V. Koeppe R.E., II Kruijtzer J.A.W. Liskamp R.M.J. de Kruijff B. Killian J.A. Biochemistry. 2001; 40: 5000-5010Crossref PubMed Scopus (156) Google Scholar) and has a hydrophobic thickness that approximately matches the hydrophobic length of the shortest peptide used (3de Planque M.R.R. Kruijtzer J.A.W. Liskamp R.M.J. Marsh D. Greathouse D.V. Koeppe R.E., II de Kruijff B. Killian J.A. J. Biol. Chem. 1999; 274: 20839-20846Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Next, we investigated the importance of Trp as interfacial anchoring residues. It has been suggested that Trp residues prefer to be positioned at a well-defined site in the lipid headgroups (3de Planque M.R.R. Kruijtzer J.A.W. Liskamp R.M.J. Marsh D. Greathouse D.V. Koeppe R.E., II de Kruijff B. Killian J.A. J. Biol. Chem. 1999; 274: 20839-20846Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 11Persson S. Killian J.A. Lindblom G. Biophys. J. 1998; 75: 1365-1371Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 12Braun P. von Heijne G. Biochemistry. 1999; 38: 9778-9782Crossref PubMed Scopus (125) Google Scholar, 13Ridder A.N. Morein S. Stam J.G. Kuhn A. de Kruijff B. Killian J.A. Biochemistry. 2000; 39: 6521-6528Crossref PubMed Scopus (116) Google Scholar, 14Yau W.M. Wimley W.C. Gawrisch K. White S.H. Biochemistry. 1998; 37: 14713-14718Crossref PubMed Scopus (815) Google Scholar, 15Wimley W.C. White S.H. Nat. Struct. Biol. 1996; 3: 842-848Crossref PubMed Scopus (1366) Google Scholar) and that thereby they can act as membrane anchors. Their abundance at the lipid/water interface in several membrane proteins (5Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A.L. Gulbis J.M. Cohen S.L. Chait B.T. Mackinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5649) Google Scholar, 6Kovacs F. Quine J. Cross T.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7910-7915Crossref PubMed Scopus (75) Google Scholar, 7Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1964) Google Scholar) is therefore likely to be functionally important, e.g. for stabilization of transmembrane helices or precise positioning of such helices at the interface. The present mass spectrometric method offers the opportunity to investigate these effects by analyzing the H/D exchange kinetics of peptides of identical total length in which the position of Trp residues along the sequence is varied. Finally, besides the length of the transmembrane helices and their interfacial anchoring behavior, the stability of the transmembrane segment is also important for membrane protein structure and function. In water-soluble proteins, the stability of the backbone of regular α-helices has been found to undergo large changes when potent breakers of both α-helical and β-sheet structures, like Pro and Gly, are inserted (e.g. see Ref. 16Eyles S.J. Gierasch L.M. J. Mol. Biol. 2000; 301: 737-747Crossref PubMed Scopus (74) Google Scholar). However, little is known about the effect on stability of these residues in transmembrane segments. Therefore, H/D exchange kinetics in transmembrane segments containing Pro and Gly residues are compared with those of peptides without these residues. The results of this study show that peptides that are long with respect to the hydrophobic thickness of the bilayer are protected from H/D exchange to a relatively large extent, which is explained in terms of induced adaptation (thickening) of the bilayer and/or a tilting of the peptides. Furthermore, the positions of the Trp residues in the transmembrane sequence are shown to be a critical factor for H/D exchange kinetics. These results suggest that Trp side chains interact strongly with the membrane/water interface at specific sites. Finally, it is shown that peptides containing Pro, but not Gly, show a markedly different H/D exchange pattern than peptides lacking this residue. This suggests a significant effect of Pro on the stability of the transmembrane α-helix. The results are discussed in relation to existing literature data on related peptide/lipid interactions. Trifluoroacetic acid was obtained from Merck (Darmstadt, Germany), 2,2,2-trifluoroethanol from Sigma. Deuterium oxide (>99.9% D) was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI). D2O was stored under nitrogen at 4 °C. Sodium iodide was from OPG Farma Company (Utrecht, The Netherlands). Ammonium acetate was from Fluka (Switzerland). The phospholipid DMPC was obtained from Avanti Polar Lipids Inc. (Birmingham, AL). The peptides WALP16, WALP19, WALP21, WALP23, WALP23inner, WALP23outer, WALP23Pro, and WALP23Gly were synthesized as described by Killianet al. (4Killian J.A. Salemink I. de Planque M.R. Lindblom G. Koeppe R.E., II Greathouse D.V. Biochemistry. 1996; 35: 1037-1045Crossref PubMed Scopus (258) Google Scholar), as modified by Greathouse et al.(17Greathouse D.V. Goforth R.L. Crawford T. van der Wel P.C.A. Killian J.A. J. Peptide Res. 2001; 57: 519-527Crossref PubMed Scopus (19) Google Scholar). The W′ALP21 peptide was synthesized as described by De Planque et al. (3de Planque M.R.R. Kruijtzer J.A.W. Liskamp R.M.J. Marsh D. Greathouse D.V. Koeppe R.E., II de Kruijff B. Killian J.A. J. Biol. Chem. 1999; 274: 20839-20846Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar) for related peptides. The peptides were tested for purity by nano-ESI-MS and found to be pure. Peptide incorporation into phospholipid vesicles was performed essentially as described previously (1Demmers J.A.A. Haverkamp J. Heck A.J.R. Koeppe R.E., II Killian J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3189-3194Crossref PubMed Scopus (103) Google Scholar). Shortly, peptides were dissolved in a small volume of trifluoroacetic acid (10 μl per mg of peptide) and dried under a nitrogen stream. To remove residual trifluoroacetic acid, the peptides were subsequently dissolved in TFE (1 mg/ml) followed by evaporation of the solvent in a rotavapor. Peptides were then again dissolved in TFE to a final concentration of 1 mg/ml. Dry mixed films of peptide and DMPC (peptide to lipid ratio of 1:25) were prepared as follows. Peptide solutions in TFE (1 ml; 0.46 mm) were added to DMPC solutions in methanol (1 ml; 12 mm) and vigorously vortexed. The solvent was removed by evaporation in a rotavapor. The mixed films were then dried for 24 h under vacuum. The films were hydrated at about 40 °C, well above the gel to liquid crystalline phase transition temperature of the phospholipid (24 °C (18Blume A. Biochemistry. 1983; 22: 5436-5442Crossref Scopus (161) Google Scholar)) in 0.5 ml of 10 mm ammonium acetate buffer (pH 7.5). Large unilamellar vesicles (LUVETs) were prepared by extrusion through a 400-nm filter at room temperature and kept at 4 °C until use. Before the start of H/D exchange, LUVETs were preincubated at 30 °C for at least 30 min. For accurate comparison of the exchange data, LUVET suspensions with different peptide composition were mixed prior to the start of the exchange. LUVET suspensions were then 50 times diluted in deuterated ammonium acetate buffer at 30 °C (10 mm, pH 7.5), containing ∼1 mm NaI. At selected time points, 2 μl of this diluted suspension was transferred into a gold-coated glass capillary and the measurement was started as quickly as possible, whereby the peptides were analyzed simultaneously. The dead-time between dilution and measurement was at a minimum 1 min. MS measurements were performed on an ESI quadrupole time-of-flight instrument (Q-Tof; Micromass Ltd., Manchester, UK) or an ESI time-of-flight instrument (LCT; Micromass Ltd., Manchester, UK), both operating in positive ion mode and equipped with a Z-spray nano-ESI source. Nano-ESI needles with a relatively large tip opening (several tens of micrometers) were prepared from borosilicate glass capillaries (Kwik-FilTM, World Precision Instruments Inc., Sarasota, Florida) on a P-97 puller (Sutter Instrument Co., Novato, CA). The needles were coated with a thin gold layer (∼500 Å) using an Edwards Scancoat sputter-coater 501 (at 40 mV, 1 kV, for 200 s). The nano-ESI capillary was positioned ∼5 mm before the orifice of the mass spectrometer. For MS experiments, the quadrupole was set in the RF only mode to act as an ion guide to efficiently link the ES ion source with the reflectron time-of-flight analyzer. The potential between the nano-ESI capillary and the orifice of the mass spectrometer was typically set to 1800 V, the cone voltage was 140 V. The nanospray needle was constantly kept at ∼30 °C. In MS/MS mode on the Q-Tof instrument, the quadrupole was used to select precursor ions, which were fragmented in the hexapole collision cell, generating product ions that were subsequently mass analyzed by the orthogonal time-of-flight mass analyzer. For MS/MS measurements on the Q-Tof instrument, the collision energy was set to 150 V. Argon was used as the collision gas. The quadrupole mass resolution parameters were set to a relatively large mass window to select the entire isotope envelope of the precursor ions. The reflectron time-of-flight parameters were set such that the fragment ions were detected at more than unit mass resolution, as required to obtain isotopically resolved H/D profiles. Increases in deuterium content (in Da) were calculated by using the average mass-to-charge (m/z) values of the isotope clusters of the undeuterated peptide and the (partly) deuterated peptides. To study effects of the hydrophobic length of transmembrane peptides on H/D exchange properties, a series of WALP/DMPC systems with varying peptide lengths (see Table I) were prepared for nano-ESI-MS measurements. To allow accurate detection of small differences in exchange properties of different peptides, all experiments were performed with mixtures of two or more peptide-lipid systems synchronically. Therefore, LUVET suspensions with different peptide composition were mixed prior to the start of the exchange. Because such combination experiments are performed under identical experimental conditions, the deuterium levels can be compared directly. Fig. 1illustrates that this method works. This figure presents, as an example, ESI mass spectra derived from simultaneously incubated and injected LUVETs of WALP21/DMPC and WALP23/DMPC dispersed in buffer, recorded by the direct proteoliposome method. The extremely hydrophobic membrane peptides are almost exclusively detected as [M+Na]+ ions, whereas the monomers, dimers, trimers, and tetramers of DMPC are observed as [M+H]+ as well as [M+Na]+ ions. The zoomed-in spectra in theinset show the isotope envelopes of the [M+Na]+ ions of the non-deuterated reconstituted WALP21 and WALP23 peptides (top), as well as those of the WALP peptides reconstituted in bilayer dispersions that were incubated for 5 min in buffered D2O (bottom). The shift in mass of the peptides indicates uptake of about 8.5–9 deuterium atoms in both cases. Moreover, the spectra indicate that the vesicles are disrupted during the ionization process in such a way that only monomers and small oligomers of phospholipid molecules are observed. The effect of increasing the hydrophobic length of the peptides with respect to the hydrophobic thickness of the bilayer was tested using a series of WALP peptides with varying hydrophobic core lengths (see Table I). Fig. 2 shows the measured deuterium content as a function of incubation time in deuterated buffer. All WALP peptides incorporated in DMPC show a fast initial exchange. Within the experimental dead-time (∼1.5 min) all peptides have taken up about 7–9 deuterium atoms. After this fast exchange the deuterium content continues to increase gradually with time, though at a much smaller exchange rate (defined as intermediate exchange rate, Ref. 1Demmers J.A.A. Haverkamp J. Heck A.J.R. Koeppe R.E., II Killian J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3189-3194Crossref PubMed Scopus (103) Google Scholar). Fig. 2 shows that all peptides have similar exchange kinetics curves in the first 20 min after incubation. All curves level off to a deuterium level, which is low as compared with the total number of labile hydrogens present in each peptide, which varies from 22 to 29 (see Table I). Furthermore, the deuterium content increases with increasing peptide length. However, the differences in deuterium levels are relatively small and represent only a fraction of the number of additional hydrogens that become available for exchange when the peptide length is increased. By fitting the kinetic curves using a non-linear squares fitting to multiexponential functions in all peptides, three kinetic regions of exchange rates representing hydrogen populations were distinguished. In the short time range from 0 to 20 min, the fast and intermediate exchangeable hydrogens exchange, whereas the remaining hydrogens exchange only after ∼20 min. The number of hydrogens in specific populations are shown in Table II. As was shown previously (1Demmers J.A.A. Haverkamp J. Heck A.J.R. Koeppe R.E., II Killian J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3189-3194Crossref PubMed Scopus (103) Google Scholar), and will also be shown later in this work, the fast exchanging hydrogens are predominantly the hydrogens at the N and C termini of the peptides, sticking out of the bilayer, while the hydrogens with intermediate exchange rates are the ones close to the Trp in the interfacial region. The remaining hydrogens, including the ones in the hydrophobic core have rate constants of (much) smaller than 0.10 min−1. These slow exchange rates are a result of both the extremely low partition coefficient of D2O in a lipid bilayer (19Jansen M. Blume A. Biophys. J. 1995; 68: 997-1008Abstract Full Text PDF PubMed Scopus (158) Google Scholar) and the involvement of the amide protons in hydrogen bonding because of α-helix formation. The amount of fast exchanged hydrogens increases with the length of the peptides but does not increase quantitatively with the numbers of exchangeable hydrogens. For instance, only about one-third of the extra number of hydrogens of WALP23 compared with WALP16 exchanges rapidly and, therefore, the majority of the extra hydrogens of WALP23 are protected against exchange.Table IINumbers of fast, intermediate, and remaining hydrogens in the investigated WALP peptidesPeptideNo. of fast HaStandard errors are below 10%.No. of intermediate HaStandard errors are below 10%.No. of remaining HaStandard errors are below 10%.WALP166.92.412.7WALP198.02.914.1WALP218.52.915.6WALP239.13.516.4The rates of exchange for the fast exchangeable hydrogens are all >1.2 min−1 for the intermediate hydrogens between 0.16 and 0.26 min−1 and for the hydrogens remaining after 20 min, smaller than 0.10 min−1.a Standard errors are below 10%. Open table in a new tab The rates of exchange for the fast exchangeable hydrogens are all >1.2 min−1 for the intermediate hydrogens between 0.16 and 0.26 min−1 and for the hydrogens remaining after 20 min, smaller than 0.10 min−1. These results may be explained by several possible mechanisms. First, the number of protected hydrogens may be higher due to a larger core region of the α-helix in the longer peptides. Second, the longer peptides may be more protected by the bilayer itself, as a result of tilting of the longer helices, or due to adaptation of the lipids to the peptide/lipid mismatch by stretching of the acyl chains (2de Planque M.R.R. Greathouse D.V. Koeppe R.E., II Schafer H. Marsh D. Killian J.A. Biochemistry. 1998; 37: 9333-9345Crossref PubMed Scopus (243) Google Scholar). These possibilities will be discussed in more detail below (see “Discussion”). In principle, an alternative possible explanation for the protection of part of the peptides against H/D exchange would be the formation of peptide aggregates, which would also hamper access of D2O molecules to the amide hydrogens. However, several observations argue against this. First, no restricted component was observed in electron spin resonance (ESR) measurements in these peptide/lipid systems, suggesting that WALP peptides are most probably present as monomeric transmembrane helices (2de Planque M.R.R. Greathouse D.V. Koeppe R.E., II Schafer H. Marsh D. Killian J.A. Biochemistry. 1998; 37: 9333-9345Crossref PubMed Scopus (243) Google Scholar). Second, nano-flow ESI-MS experiments on the non-soluble WALP23 in buffer showed that when this dispersion was injected, not even a trace amount of WALP23 could be detected after extensive sonication (data not shown). Remarkably, after addition of TFE (1:1; volume ratio), which presumably breaks up the aggregates, the peptide could again be easily detected in the spectrum. It is therefore concluded that it is not possible to measure aggregated peptides directly by ESI-MS, indicating that the WALP peptides of which the mass spectra are analyzed in the present study do not originate from aggregates. It has been proposed that Trp residues have strong interactions with the interface. To gain more insight into the potential anchoring role of the Trp residues, the positions of this aromatic amino acid were varied in peptides of invariable length. Moreover, to make the putative interaction zone less broad, the number of Trp residues was reduced to one at each terminus of the peptide (see Table I). WALP23inner has Trp residues at the 3 and 21 positions, whereas WALP23outer has Trp residues at positions 2 and 22. Fig. 3shows the deuterium incorporation of both peptides reconstituted in DMPC after synchronic dilution in deuterated buffer and subsequent mass spectrometric analysis. Although both peptides have the same number of exchangeable hydrogens, they clearly show different H/D exchange behavior. The deuterium level curve for WALP23inner lies well above the one of WALP23outer, both in the short term region as well as after longer incubation periods. Comparing the averagem/z values of the isotope clusters of the peptides in both undeuterated and deuterated buffer results in a 1.6 (± 0.2) Da higher deuterium content for WALP23inner. This suggests that WALP23inner has a larger population of fast exchangeable hydrogens. Consequently, WALP23inner incorporated in a DMPC bilayer seems to have a shorter protected region that could be due to a shorter inter-Trp distance. The deuteration levels of WALP23inner are also on the long term about 2 Da higher than for WALP23outer. These results suggest that the position of the Trp side chain determines the extent of protection in the bilayer environment, consistent with the idea that Trp forms a strong interfacial anchor (3de Planque M.R.R. Kruijtzer J.A.W. Liskamp R.M.J. Marsh D. Greathouse D.V. Koeppe R.E., II de Kruijff B. Killian J.A. J. Biol. Chem. 1999; 274: 20839-20846Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 12Braun P. von Heijne G. Biochemistry. 1999; 38: 9778-9782Crossref PubMed Scopus (125) Google Scholar, 14Yau W.M. Wimley W.C. Gawrisch K. White S.H. Biochemistry. 1998; 37: 14713-14718Crossref PubMed Scopus (815) Google Scholar, 15Wimley W.C. White S.H. Nat. Struct. Biol. 1996; 3: 842-848Crossref PubMed Scopus (1366) Google Scholar). The time course for exchange of WALP23inner appears to match that of WALP23, when comparing the data in Figs. 2 and 3. However, if one assumes that the tryptophan side chain hydrogens exchange fast, then WALP23inner and WALP23outer would have two fast exchangeable hydrogens less than WALP23, and therefore the data would suggest that it is the backbone exchange of WALP23outer that matches well that of WALP23. Measurements with W′ALP21, which contains methylated Trp residues (see Table I), and thus has no exchangeable hydrogen atom in the indole side chain moiety, suggested that these indole hydrogens indeed exchange rapidly. It was found that the H/D exchange curve of this peptide lies close to 4 Da lower than that of WALP21 after both short and longer incubation times (data not shown). The apparent similarity in backbone exchange kinetics of WALP23 and WALP23outer is consistent with the idea that in the unfavorable case when the peptide is long with respect to the h" @default.
• W2029159154 created "2016-06-24" @default.
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• W2029159154 date "2001-09-01" @default.
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• W2029159154 title "Interfacial Positioning and Stability of Transmembrane Peptides in Lipid Bilayers Studied by Combining Hydrogen/Deuterium Exchange and Mass Spectrometry" @default.
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