FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023388639> ?p ?o ?g. }

• W2023388639 endingPage "18396" @default.
• W2023388639 startingPage "18388" @default.
• W2023388639 abstract "Model peptides composed of alanine and leucine residues are often used to mimic single helical transmembrane domains. Many studies have been carried out to determine how they interact with membranes. However, few studies have investigated their lipid-destabilizing effect. We designed three peptides designated KALRs containing a hydrophobic stretch of 14, 18, or 22 alanines/leucines surrounded by charged amino acids. Molecular modeling simulations in an implicit membrane model as well as attenuated total reflection-Fourier transform infrared analyses show that KALR is a good model of a transmembrane helix. However, tryptophan fluorescence and attenuated total reflection-Fourier transform infrared spectroscopy indicate that the extent of binding and insertion into lipids increases with the length of the peptide hydrophobic core. Although binding can be directly correlated to peptide hydrophobicity, we show that insertion of peptides into a membrane is determined by the length of the peptide hydrophobic core. Functional studies were performed by measuring the ability of peptides to induce lipid mixing and leakage of liposomes. The data reveal that whereas KALR14 does not destabilize liposomal membranes, KALR18 and KALR22 induce 40 and 50% of lipid-mixing, and 65 and 80% of leakage, respectively. These results indicate that a transmembrane model peptide can induce liposome fusion in vitro if it is long enough. The reasons for the link between length and fusogenicity are discussed in relation to studies of transmembrane domains of viral fusion proteins. We propose that fusogenicity depends not only on peptide insertion but also on the ability of peptides to destabilize the two leaflets of the liposome membrane. Model peptides composed of alanine and leucine residues are often used to mimic single helical transmembrane domains. Many studies have been carried out to determine how they interact with membranes. However, few studies have investigated their lipid-destabilizing effect. We designed three peptides designated KALRs containing a hydrophobic stretch of 14, 18, or 22 alanines/leucines surrounded by charged amino acids. Molecular modeling simulations in an implicit membrane model as well as attenuated total reflection-Fourier transform infrared analyses show that KALR is a good model of a transmembrane helix. However, tryptophan fluorescence and attenuated total reflection-Fourier transform infrared spectroscopy indicate that the extent of binding and insertion into lipids increases with the length of the peptide hydrophobic core. Although binding can be directly correlated to peptide hydrophobicity, we show that insertion of peptides into a membrane is determined by the length of the peptide hydrophobic core. Functional studies were performed by measuring the ability of peptides to induce lipid mixing and leakage of liposomes. The data reveal that whereas KALR14 does not destabilize liposomal membranes, KALR18 and KALR22 induce 40 and 50% of lipid-mixing, and 65 and 80% of leakage, respectively. These results indicate that a transmembrane model peptide can induce liposome fusion in vitro if it is long enough. The reasons for the link between length and fusogenicity are discussed in relation to studies of transmembrane domains of viral fusion proteins. We propose that fusogenicity depends not only on peptide insertion but also on the ability of peptides to destabilize the two leaflets of the liposome membrane. Biological membranes are a complex mixture of lipids that contain proteins, hydrocarbons, and other constituents (1Hauser H. Poupart G. Yeagle P.L. The Structure of Biological Membranes. 2nd Ed. CRC Press LLC, Boca Raton, FL2005Google Scholar). In addition, expression, folding, and insolubility problems can make the study of protein-membrane interactions very complicated and limit the results of interpretation (2Eiler S. Gangloff M. Duclaud S. Moras D. Ruff M. Protein Expression Purif. 2001; 22: 165-173Crossref PubMed Scopus (83) Google Scholar, 3Nakayama M. Ohara O. Biochem. Biophys. Res. Commun. 2003; 312: 825-830Crossref PubMed Scopus (19) Google Scholar). Therefore, peptide-membrane interactions are often studied with liposomes of strictly controlled composition (4Martin I. Ruysschaert J.M. Biosci. Rep. 2000; 20: 483-500Crossref PubMed Scopus (41) Google Scholar, 5Busquets M.A. Alsina M.A. Haro I. Curr. Drug Targets. 2003; 4: 633-642Crossref PubMed Scopus (7) Google Scholar, 6Weiss T.M. van der Wel P.C.A. Killian J.A. Koeppe R.E. Huang H.W. Biophys. J. 2003; 84: 379-385Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 7Lorin A. Thomas A. Stroobant V. Brasseur R. Lins L. Chem. Phys. Lipids. 2006; 141: 185-196Crossref PubMed Scopus (9) Google Scholar, 8Charloteaux B. Lorin A. Crowet J.M. Stroobant V. Lins L. Thomas A. Brasseur R. J. Mol. Biol. 2006; 359: 597-609Crossref PubMed Scopus (22) Google Scholar). Use of such model membrane systems also allows one to control the surrounding medium (4Martin I. Ruysschaert J.M. Biosci. Rep. 2000; 20: 483-500Crossref PubMed Scopus (41) Google Scholar). The same principle has been used by several groups with simplified de novo model peptides composed of typical residues (9Davis J.H. Clare D.M. Hodges R.S. Bloom M. Biochemistry. 1983; 22: 5298-5305Crossref Scopus (122) Google Scholar, 10Bechinger B. J. Mol. Biol. 1996; 263: 768-775Crossref PubMed Scopus (172) Google Scholar, 11Bechinger B. Biophys. J. 2001; 81: 2251-2256Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 12Zhao J.B. Kimura S. Imanishi Y. Biochim. Biophys. Acta. 1996; 1283: 37-44Crossref PubMed Scopus (5) Google Scholar, 13Liu L.P. Li S.C. Goto N.K. Deber C.M. Biopolymers. 1996; 39: 465-470Crossref PubMed Scopus (68) Google Scholar, 14Killian J.A. Salemink I. de Planque M.R.R. Lindblom G. Koeppe R.E. Greathouse D.V. Biochemistry. 1996; 35: 1037-1045Crossref PubMed Scopus (262) Google Scholar, 15Lewis R.N.A.H. Zhang Y.P. Hodges R.S. Subczynski W.K. Kusumi A. Flach C.R. Mendelsohn R. McElhaney R.N. Biochemistry. 2001; 40: 12103-12111Crossref PubMed Scopus (35) Google Scholar, 16de Planque M.R.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (259) Google Scholar, 17Ozdirekcan S. Rijkers D.T.S. Liskamp R.M.J. Killian J.A. Biochemistry. 2005; 44: 1004-1012Crossref PubMed Scopus (88) Google Scholar). These approaches are useful for analyzing the impact of specific features of peptides and/or membranes on peptide-membrane interactions (12Zhao J.B. Kimura S. Imanishi Y. Biochim. Biophys. Acta. 1996; 1283: 37-44Crossref PubMed Scopus (5) Google Scholar, 18Killian J.A. FEBS Lett. 2003; 555: 134-138Crossref PubMed Scopus (135) Google Scholar).The effect of hydrophobicity of TM 6The abbreviations used are: TM, transmembrane; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPG, 1,2-dioleoyl-sn-glycero-3-phospho-racemic-(1-glycerol); DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; CHOL, cholesterol; SM, sphingomyelin; LUV, large unilamellar vesicle; SUV, small unilamellar vesicle; FTIR, Fourier transform infrared; ATR, attenuated total reflection; TFE, trifluoroethanol; DPX, N,N′-p-xylenebis (pyridinium bromide); HPTS, 8-hydroxypyrene-1,3,6-trisulfonic acid; IMPALA, integral membrane protein and lipid association. 6The abbreviations used are: TM, transmembrane; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPG, 1,2-dioleoyl-sn-glycero-3-phospho-racemic-(1-glycerol); DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; CHOL, cholesterol; SM, sphingomyelin; LUV, large unilamellar vesicle; SUV, small unilamellar vesicle; FTIR, Fourier transform infrared; ATR, attenuated total reflection; TFE, trifluoroethanol; DPX, N,N′-p-xylenebis (pyridinium bromide); HPTS, 8-hydroxypyrene-1,3,6-trisulfonic acid; IMPALA, integral membrane protein and lipid association. model peptides on their interaction with membranes has been studied by several groups (16de Planque M.R.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (259) Google Scholar, 19Deber C.M. Liu L.P. Wang C. Goto N.K. Reithmeier R.A.F. Curr. Top. Membr. 2002; 52: 465-479Crossref Google Scholar). A threshold of hydrophobicity is required for peptides to be able to insert into membranes and to adopt a transmembrane orientation (19Deber C.M. Liu L.P. Wang C. Goto N.K. Reithmeier R.A.F. Curr. Top. Membr. 2002; 52: 465-479Crossref Google Scholar). For example, peptides with a hydrophobic core composed of 24 alanine residues do not adopt a stable transmembrane orientation in phosphatidylcholine-hydrated membranes (15Lewis R.N.A.H. Zhang Y.P. Hodges R.S. Subczynski W.K. Kusumi A. Flach C.R. Mendelsohn R. McElhaney R.N. Biochemistry. 2001; 40: 12103-12111Crossref PubMed Scopus (35) Google Scholar). Lewis et al. concluded that this is due to insufficient hydrophobicity rather than poor helicity (15Lewis R.N.A.H. Zhang Y.P. Hodges R.S. Subczynski W.K. Kusumi A. Flach C.R. Mendelsohn R. McElhaney R.N. Biochemistry. 2001; 40: 12103-12111Crossref PubMed Scopus (35) Google Scholar). As a result, TM model peptides are usually composed of a mixture of alanine and leucine residues (16de Planque M.R.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (259) Google Scholar, 20Rinia H.A. Boots J.-W.P. Rijkers D.T.S. Kik R.A. Snel M.M.E. Demel R.A. Killian J.A. Van der E erden J.P.J.M. De Kruijff B. Biochemistry. 2002; 41: 2814-2824Crossref PubMed Scopus (77) Google Scholar, 21Chung L.A. Thompson T.E. Biochemistry. 1996; 35: 11343-11354Crossref PubMed Scopus (34) Google Scholar, 22Percot A. Zhu X.X. Lafleur M. Biopolymers. 1999; 50: 647-655Crossref PubMed Scopus (17) Google Scholar, 23Harzer U. Bechinger B. Biochemistry. 2000; 39: 13106-13114Crossref PubMed Scopus (123) Google Scholar, 24Lewis R.N.A.H. Zhang Y.P. Liu F. McElhaney R.N. Bioelectrochemistry. 2002; 56: 135-140Crossref PubMed Scopus (16) Google Scholar, 25Sharpe S. Barber K.R. Grant C.W.M. Goodyear D. Morrow M.R. Biophys. J. 2002; 83: 345-358Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 26Strandberg E. Ozdirekcan S. Rijkers D.T.S. van der Wel P.C.A. Koeppe R.E. Liskamp R.M.J. Killian J.A. Biophys. J. 2004; 86: 3709-3721Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 27Sparr E. Ash W.L. Nazarov P.V. Rijkers D.T.S. Hemminga M.A. Tieleman D.P. Killian J.A. J. Biol. Chem. 2005; 280: 39324-39331Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 28Siegel D.P. Cherezov V. Greathouse D.V. Koeppe II, R.V. Killian J.A. Caffrey M. Biophys. J. 2006; 90: 200-211Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 29Killian J.A. Nyholm T.K.M. Curr. Opin. Struct. Biol. 2006; 16: 473-479Crossref PubMed Scopus (132) Google Scholar). This composition better mimics the mean hydrophobicity of natural TM domains than peptides composed of solely leucine residues (16de Planque M.R.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (259) Google Scholar, 30Zhang Y.P. Lewis R.N.A.H. Henry G.D. Sykes B.D. Hodges R.S. McElhaney R.N. Biochemistry. 1995; 34: 2348-2361Crossref PubMed Scopus (109) Google Scholar). Almost all de novo TM peptides have aromatic or positively charged residues on either side of the hydrophobic stretch. Lysine and arginine residues are used, since they help to solubilize the peptide, to promote the monomer form, to favor a helical conformation, and to ensure the TM orientation (16de Planque M.R.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (259) Google Scholar, 22Percot A. Zhu X.X. Lafleur M. Biopolymers. 1999; 50: 647-655Crossref PubMed Scopus (17) Google Scholar). Moreover, it was shown that natural transmembrane α-helices domains are often flanked by lysine and arginine residues (31Reithmeier R.A.F. Curr. Opin. Struct. Biol. 1995; 5: 491-500Crossref PubMed Scopus (143) Google Scholar, 32Vonheijne G. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 167-192Crossref PubMed Scopus (255) Google Scholar).When the length of the hydrophobic stretch of a helical peptide fits the thickness of the membrane hydrophobic core, the peptide inserts into the membrane and adopts a TM orientation (26Strandberg E. Ozdirekcan S. Rijkers D.T.S. van der Wel P.C.A. Koeppe R.E. Liskamp R.M.J. Killian J.A. Biophys. J. 2004; 86: 3709-3721Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 33de Planque M.R.R. Boots J.W.P. Rijkers D.T.S. Liskamp R.M.J. Greathouse D.V. Killian J.A. Biochemistry. 2002; 41: 8396-8404Crossref PubMed Scopus (87) Google Scholar). When it does not fit, either polar residues are exposed to the apolar medium, or hydrophobic residues are accessible to the water phase. This unfavorable phenomenon has been called hydrophobic mismatch (6Weiss T.M. van der Wel P.C.A. Killian J.A. Koeppe R.E. Huang H.W. Biophys. J. 2003; 84: 379-385Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 16de Planque M.R.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (259) Google Scholar, 18Killian J.A. FEBS Lett. 2003; 555: 134-138Crossref PubMed Scopus (135) Google Scholar, 34Ren J.H. Lew S. Wang Z.W. London E. Biochemistry. 1997; 36: 10213-10220Crossref PubMed Scopus (185) Google Scholar). Positive and negative hydrophobic mismatches occur when the hydrophobic stretch of a peptide is too long or too short with respect to the membrane thickness, respectively (16de Planque M.R.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (259) Google Scholar). The peptide can adapt its structure or its orientation to overcome mismatch (23Harzer U. Bechinger B. Biochemistry. 2000; 39: 13106-13114Crossref PubMed Scopus (123) Google Scholar, 25Sharpe S. Barber K.R. Grant C.W.M. Goodyear D. Morrow M.R. Biophys. J. 2002; 83: 345-358Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 35de Planque M.R.R. Goormaghtigh E. Greathouse D.V. Koeppe R.E. Kruijtzer J.A.W. Liskamp R.M.J. de Kruijff B. Killian J.A. Biochemistry. 2001; 40: 5000-5010Crossref PubMed Scopus (156) Google Scholar). It can also oligomerize to reduce the mismatch (for a review, see Ref. 16de Planque M.R.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (259) Google Scholar). If the mismatch is too great, insertion of the peptide into the membrane is reduced (16de Planque M.R.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (259) Google Scholar, 34Ren J.H. Lew S. Wang Z.W. London E. Biochemistry. 1997; 36: 10213-10220Crossref PubMed Scopus (185) Google Scholar, 35de Planque M.R.R. Goormaghtigh E. Greathouse D.V. Koeppe R.E. Kruijtzer J.A.W. Liskamp R.M.J. de Kruijff B. Killian J.A. Biochemistry. 2001; 40: 5000-5010Crossref PubMed Scopus (156) Google Scholar, 36Moll T.S. Thompson T.E. Biochemistry. 1994; 33: 15469-15482Crossref PubMed Scopus (42) Google Scholar, 37Webb R.J. East J.M. Sharma R.P. Lee A.G. Biochemistry. 1998; 37: 673-679Crossref PubMed Scopus (132) Google Scholar). It has been shown that membrane adaptation can also result as a response to a hydrophobic mismatch. 2H NMR measurements with lipids containing a perdeuterated acyl chain, x-ray diffraction, and differential scanning calorimetry measurements revealed that a positive mismatch increases the lipid chain order, whereas a negative mismatch increases membrane disorder (38Zhang Y.P. Lewis R.N.A.H. Hodges R.S. McElhaney R.N. Biochemistry. 1992; 31: 11579-11588Crossref PubMed Scopus (157) Google Scholar, 39de Planque M.R.R. Kruijtzer J.A.W. Liskamp R.M.J. Marsh D. Greathouse D.V. Koeppe R.E. de Kruijff B. Killian J.A. J. Biol. Chem. 1999; 274: 20839-20846Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 40Morein S. Strandberg E. Killian J.A. Persson S. Arvidson G. Koeppe R.E. Lindblom G. Biophys. J. 1997; 73: 3078-3088Abstract Full Text PDF PubMed Scopus (60) Google Scholar, 41Morein S. Killian J.A. Sperotto M.M. Biophys. J. 2002; 82: 1405-1417Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). However, other studies using 15N as well as 31P NMR, differential scanning calorimetry, and ESR showed that peptides long enough or too long to span the membrane perturb the membrane order whereas peptides that are too short have less effect (23Harzer U. Bechinger B. Biochemistry. 2000; 39: 13106-13114Crossref PubMed Scopus (123) Google Scholar, 24Lewis R.N.A.H. Zhang Y.P. Liu F. McElhaney R.N. Bioelectrochemistry. 2002; 56: 135-140Crossref PubMed Scopus (16) Google Scholar).Few studies have focused on the ability of TM model peptides to induce membrane fusion. Hofmann et al. (42Hofmann M.W. Weise K. Ollesch J. Agrawal P. Stalz H. Stelzer W. Hulsbergen F. de Groot H. Gerwert K. Reed J. Langosch D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14776-14781Crossref PubMed Scopus (76) Google Scholar) showed that the fusogenicity of TM model peptides varies with the ratio of helix-promoting leucine and sheet-promoting valine residues and is enhanced if helix-destabilizing residues, such as glycine and proline, are present within their hydrophobic core. They further showed that fusogenicity of these peptides correlates with structural flexibility (42Hofmann M.W. Weise K. Ollesch J. Agrawal P. Stalz H. Stelzer W. Hulsbergen F. de Groot H. Gerwert K. Reed J. Langosch D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14776-14781Crossref PubMed Scopus (76) Google Scholar). The more flexible the peptide is, the more fusion is induced.In this study, we analyzed the interaction of de novo TM peptides with membranes and in particular their ability to induce fusion. We designed three TM model peptides; all have a hydrophobic core made of alanines and leucines (14, 18, and 22 residues), and both the N and C termini were extended with three positively charged residues. Interactions and fusogenic properties of each KALR with a membrane were studied by modeling and experimental approaches.EXPERIMENTAL PROCEDURESMaterials—1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-racemic-(1-glycerol) sodium salt (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (CHOL), and bovine brain sphingomyelin (SM) were purchased from Sigma. Trifluoroethanol (TFE), Hepes, Triton X-100, and Me2SO were purchased from Sigma. Octadecylrhodamine chloride B, N,N′-p-xylenebis(pyridinium bromide) (DPX), and 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) came from Molecular Probes, Inc. (Eugene, OR). NaCl came from Merck Eurolab (Leuven, Belgium). Peptides KALR14, KALR18, and KALR22 were synthesized by NeoMPS (Strasbourg, France). These peptides have free N and C termini (Table 1). Their purity is higher than 85%.TABLE 1Sequence of KALR peptidesPeptideSequenceKALR14KKKAALAAALWAALAAARRRKALR18KKKAALAAALAAWLAAALAAARRRKALR22KKKAALAAALAAALWAALAAALAAARRR Open table in a new tab Liposome Preparation—DOPC, DOPG, DOPE, SM, and CHOL were dissolved in a chloroform/methanol (2:1, v/v) solution. DOPC/CHOL/DOPE/DOPG/SM (34:33:16:10:7 mol/mol/mol/mol/mol) film was obtained after evaporation under vacuum with a rotary evaporator (Rotovapor R-3000, Van Der Heyden Büchi, Switzerland). The lipid film, dried for one night, was then dispersed in 2 ml of 5 mm HEPES and 100 mm NaCl, pH 7.4, buffer and incubated for 1 h at 37 °C.To obtain DOPC/CHOL/DOPE/DOPG/SM (34:33:16:10:7, mol/mol/mol/mol/mol) large unilamellar vesicles (LUV), the hydrated lipid dispersion was exposed to five freeze-thaw cycles and passed 10 times through a polycarbonate membrane (0.1 μm) under 20 bars pressure with an extruder (Lipex Biomembranes, Vancouver, Canada). To obtain DOPC/CHOL/DOPE/DOPG/SM (34/33/16/10/7 mol/mol/mol/mol/mol) small unilamellar vesicles (SUV), the hydrated film was sonicated (high intensity ultrasonic processor; Sigma) for 5 min at 50 W. A 10,000 × g centrifugation for 2 min (Biofuge Pico, Van der Heyden, Heraeus, Germany) discarded titanium deposit and residual multilamellar vesicles. The phospholipid concentration was determined by phosphorus analysis (43Mrsny R.J. Volwerk J.J. Griffith O.H. Chem. Phys. Lipids. 1986; 39: 185-191Crossref PubMed Scopus (97) Google Scholar).Lipid-binding Experiments—In a polar environment, the tryptophan present in KALRs has a fluorescence spectrum with a maximum around 350 nm. Upon the addition of SUVs, the fluorescence maximum shifts to the blue, around 335 nm. The affinity of the peptide for the SUV was determined by adding increasing amounts of vesicles to 0.67 mm peptides dissolved in Me2SO, as previously described (44Schwarz G. Stankowski S. Rizzo V. Biochim. Biophys. Acta. 1986; 861: 141-151Crossref PubMed Scopus (162) Google Scholar, 45Rizzo V. Stankowski S. Schwarz G. Biochemistry. 1987; 28: 2751-2759Crossref Scopus (158) Google Scholar, 46Lear J.D. DeGrado W.F. J. Biol. Chem. 1987; 262: 6500-6505Abstract Full Text PDF PubMed Google Scholar). Fluorescence was recorded at room temperature (λexc, 280 nm; λem, 335 nm) after each addition on an LS-50B PerkinElmer Life Sciences fluorimeter. The fluorescence values were then corrected by taking into account the dilution factor corresponding to the addition of the liposomes and by subtracting the corresponding blank (Me2SO with the same amount of SUVs). The ratio (R) of bound to total peptides and the dissociation constant (Kd) were calculated as described by Lear and DeGrado (46Lear J.D. DeGrado W.F. J. Biol. Chem. 1987; 262: 6500-6505Abstract Full Text PDF PubMed Google Scholar). Experiments were carried out in a buffer composed of 5 mm HEPES and 100 mm NaCl at pH 7.4.Lipid Mixing Experiments—Mixing of liposome membranes was followed by measuring the fluorescence increase of octadecylrhodamine chloride B, a lipid soluble probe, after the fusion of labeled and unlabeled liposomes. Labeled liposomes were obtained by incorporating octadecylrhodamine chloride B in the dry lipid film at a 5% concentration of the total lipid weight. Labeled and unlabeled liposomes were mixed at a weight ratio of 1:4, respectively, and at a final concentration of 12.5 μm in 5 mm HEPES and 100 mm of NaCl buffer at pH 7.4. 100% of fusion was determined by adding Triton X-100 at 2% to labeled/unlabeled (1:4) LUVs. Fluorescence was recorded at room temperature (λexc, 560 nm; λem, 590 nm) on an LS-50B PerkinElmer Life Sciences fluorimeter. The tests were performed with LUVs and were repeated three times with different batches of peptide.Leakage of Liposome Vesicle Contents—Vesicle leakage was monitored using an assay based on the quenching of HPTS by DPX (47Van Bambeke F. Kerkhofs A. Schanck A. Remacle C. Sonveaux E. Tulkens P.M. Mingeot-Leclerq M.P. Lipids. 2000; 35: 213-223Crossref PubMed Google Scholar). HPTS and DPX are both encapsulated in the aqueous phase of the same liposomes. Leakage of vesicles was followed by measuring the dequenching of HPTS released into the medium. Fluorescence was recorded at room temperature (λexc, 450 nm; λem, 512 nm) on an LS-50B PerkinElmer fluorimeter. Liposomes (LUVs) were prepared as described above in 12.5 mm HPTS (45 mm NaCl), 45 mm DPX (20 mm NaCl), and 10 mm HEPES buffer at pH 7.4. Vesicles containing encapsulated HPTS and DPX were eluted in the void volume of a Sephadex G-75 column, with 5 mm HEPES and 100 mm NaCl buffer (pH 7.4). Assays were repeated three times with different batches of peptide.Electron Microscopy—The effect of the peptides on SUV was examined by negative transmission staining electron microscopy. Prior to staining and fixing, suspensions of SUV at 4.5 mm (with or without lyophilized peptide) were incubated for 5 min at room temperature. A drop containing SUV alone or with a peptide was deposited onto a carbon-coated grid and negatively stained with 2% phosphotungstic acid (pH 6.8). The grids were observed by using a JEOL JEM 100B electron microscope (Japan Electron Optics Laboratory Co., Tokyo, Japan).IR Spectroscopy Measurements—Spectra were recorded at room temperature on a Brüker Equinox 55 equipped with a liquid nitrogen-cooled mercury-cadmium-telluride detector at a resolution of 2 cm-1 by averaging 512 scans. Reference spectra of the germanium plate were automatically recorded after purging for 15 min with dry air and subtracted from the recently run sample spectrum. The plate was sealed in a universal sample holder and rehydrated by flushing the holder with N2 saturated with D2O for 3 h at room temperature. Free peptide samples (50 μg of peptide in TFE) and the lipid-bound peptides were spread out on a germanium ATR plate (50 × 20 × 2 mm; Aldrich Chimica) with an aperture of 45°, yielding 25 internal reflections. The lipid-membrane sample was prepared by incubation of 1 ml of peptides at 60 μm with 6 ml of CHOL/DOPC/DOPE/DOPG (40/34/16/10, mol/mol/mol/mol/mol) SUV at 500 μm (1:50 peptide/lipid (mol/mol) ratio) in 5 mm HEPES and 100 mm NaCl buffer at pH 7.4 for 1 h at room temperature. After incubation, the lipid/peptide mixture was filtered through an anisotropic hydrophilic YM 30 membrane (cut-off 30 kDa) of a Centrifree micropartition system (Amicon) with a 13,000 × g centrifugation for 45 min to separate lipid-associated from free peptides. Distilled water was used to recuperate the retained sample containing the peptide-lipid complex. To determine the peptide insertion rate in membranes by infrared spectra, the area of the amide I peak was divided by the area of the lipid C=O peak and the number of amide groups in the peptide, as previously shown (35de Planque M.R.R. Goormaghtigh E. Greathouse D.V. Koeppe R.E. Kruijtzer J.A.W. Liskamp R.M.J. de Kruijff B. Killian J.A. Biochemistry. 2001; 40: 5000-5010Crossref PubMed Scopus (156) Google Scholar).To evaluate the orientation of peptides in lipids, spectra were recorded at two orthogonal linear polarizations (90 and 0°) of the incident light. The dichroic spectrum was obtained by subtracting the spectrum recorded with polarized light at 0° from that at 90°. The angle between the germanium crystal and the dipole was calculated from the dichroic ratio RATR/RATR = A(90°)/A(0°), where A(90°) is the absorbance of the selected dipole from a spectrum recorded with polarized light at 90°, and A(0°) is the absorbance of the same dipole from a 0° polarized spectrum. The bands chosen to characterize the protein and phospholipid orientation are the amide I and the lipid νs (CH2), respectively (48Goormaghtigh E. Raussens V. Ruysschaert J.M. Biochim. Biophys. Acta. 1999; 1422: 105-185Crossref PubMed Scopus (506) Google Scholar, 49Castano S. Desbat B. Biochim. Biophys. Acta. 2005; 1715: 81-95Crossref PubMed Scopus (85) Google Scholar).Design of KALRs—Three peptides with a hydrophobic core of 14, 18, and 22 residues were designed (Table 1). The hydrophobic core is an AA(LAAA)n motif with n = 3, 4, and 5 for KALR14, KALR18, and KALR22, respectively. The hydrophobic core lengths were chosen to have variations of approximately one helix turn, corresponding to 6 Å (50Creighton T.E. Proteins: Structures and Molecular Properties. 2nd ed. W. H. Freeman and Co., New York1992: 182-186Google Scholar). Each peptide is surrounded by three lysine residues at the N-terminal extremity and three arginine residues at the C-terminal extremity. At the center of the sequence of each peptide, a tryptophan residue replaces an alanine residue in order to have a fluorescent sensor.Analysis by Molecular Modeling—The integral membrane protein and lipid association (IMPALA) method was used to analyze interaction of KALRs with membranes (51Ducarme P. Rahman M. Brasseur R. Proteins. 1998; 30: 357-371Crossref PubMed Scopus (102) Google Scholar). The thickness of the implicit bilayer was extrapolated from experimental data on DOPC bilayers (16de Planque M.R.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (259) Google Scholar, 52Nagle J.F. Tristram-Nagle S. Biochim. Biophys. Acta. 2000; 1469: 159-195Crossref PubMed Scopus (2207) Google Scholar, 53Kucerka N. Tristram-Nagle S. Nagle J.F. J. Membr. Biol. 2005; 208: 193-202Crossref PubMed Scopus (618) Google Scholar) and the contribution of SM/cholesterol to membrane thickness (54Nezil F.A. Bloom M. Biophys. J. 1992; 61: 1176-1183Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 55Cantor R.S. Biophys. J. 1999; 76: 2625-2639Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 56Simons K. Vaz W.L.C. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 269-295Crossref PubMed Scopus (1342) Google Scholar, 57Salamon Z. Devanathan S. Alves I.D. Tollin G. J. Biol. Chem. 2005; 280: 11175-11184Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The thickness of the model membrane was 40 Å with a hydrophobic core of 31 Å to mimic membranes used in the experimental assays.The IMPALA method simulates the insertion of peptides into the bilayer by adding energy restraint functions to the usual energy function of peptides. The lipid bilayer is defined by C(z), which represents an empirical function describing membrane properties as follows, C(Z)=1-11+eα(z-z0)(Eq. 1) where z is perpendicular to the membrane and has its origin at the center of the bilayer. The values α and z0 are parameters fixed in such a way that C(|z ≥ 20 Å|) = 1 and C(|z ≤ 15.5 Å|) = 0. The value of the function is constant from - ∞ to -20 Å (hydrophilic phase), from -15.5 to 15.5 Å (hydrophobic core), and from 20 Å to ∞ (hydrophilic phase).Two restraints simulate the interaction between the peptide and the bilayer. The first one accounts for the effect that pushes hydrophobic atoms into the membrane (hydrophobic restraint) and hydrophilic atoms outside of it as follows, Epho=-∑i=1Ns(i)Etr(i)C(zi)(Eq. 2) where N is the total number of atoms, S(i) is the solvent-accessible surface of atom i, Etr(i) is its transfer energy in units of accessible surface area, and C(zi) is the value of C(z) at the position zi of a" @default.
• W2023388639 created "2016-06-24" @default.
• W2023388639 creator A5014511259 @default.
• W2023388639 creator A5050533193 @default.
• W2023388639 creator A5055176983 @default.
• W2023388639 creator A5068262373 @default.
• W2023388639 creator A5072323087 @default.
• W2023388639 creator A5077610815 @default.
• W2023388639 date "2007-06-01" @default.
• W2023388639 modified "2023-10-13" @default.
• W2023388639 title "Mode of Membrane Interaction and Fusogenic Properties of a de Novo Transmembrane Model Peptide Depend on the Length of the Hydrophobic Core" @default.
• W2023388639 cites W1517533244 @default.
• W2023388639 cites W1518606102 @default.
• W2023388639 cites W1535301196 @default.
• W2023388639 cites W1582112214 @default.
• W2023388639 cites W1814801478 @default.
• W2023388639 cites W1968028474 @default.
• W2023388639 cites W1968301751 @default.
• W2023388639 cites W1970276965 @default.
• W2023388639 cites W1972159702 @default.
• W2023388639 cites W1973918451 @default.
• W2023388639 cites W1974749892 @default.
• W2023388639 cites W1976747271 @default.
• W2023388639 cites W1979411809 @default.
• W2023388639 cites W1980744846 @default.
• W2023388639 cites W1982529725 @default.
• W2023388639 cites W1983718883 @default.
• W2023388639 cites W1986324420 @default.
• W2023388639 cites W1987739907 @default.
• W2023388639 cites W1990136319 @default.
• W2023388639 cites W1991743475 @default.
• W2023388639 cites W1992909182 @default.
• W2023388639 cites W1997279293 @default.
• W2023388639 cites W1999077168 @default.
• W2023388639 cites W1999859501 @default.
• W2023388639 cites W2015258557 @default.
• W2023388639 cites W2016343967 @default.
• W2023388639 cites W2016989448 @default.
• W2023388639 cites W2021966802 @default.
• W2023388639 cites W2023100445 @default.
• W2023388639 cites W2026088739 @default.
• W2023388639 cites W2030210434 @default.
• W2023388639 cites W2031384239 @default.
• W2023388639 cites W2031408917 @default.
• W2023388639 cites W2032966118 @default.
• W2023388639 cites W2035035273 @default.
• W2023388639 cites W2035664036 @default.
• W2023388639 cites W2040255016 @default.
• W2023388639 cites W2040309290 @default.
• W2023388639 cites W2040333135 @default.
• W2023388639 cites W2040585305 @default.
• W2023388639 cites W2040727080 @default.
• W2023388639 cites W2042249476 @default.
• W2023388639 cites W2042360395 @default.
• W2023388639 cites W2042595932 @default.
• W2023388639 cites W2042968365 @default.
• W2023388639 cites W2046305383 @default.
• W2023388639 cites W2048623371 @default.
• W2023388639 cites W2055592362 @default.
• W2023388639 cites W2057108047 @default.
• W2023388639 cites W2059448452 @default.
• W2023388639 cites W2060878281 @default.
• W2023388639 cites W2066020219 @default.
• W2023388639 cites W2068923789 @default.
• W2023388639 cites W2071914336 @default.
• W2023388639 cites W2072757581 @default.
• W2023388639 cites W2075836002 @default.
• W2023388639 cites W2077471821 @default.
• W2023388639 cites W2084102523 @default.
• W2023388639 cites W2085242657 @default.
• W2023388639 cites W2086700428 @default.
• W2023388639 cites W2092111186 @default.
• W2023388639 cites W2094170312 @default.
• W2023388639 cites W2096254207 @default.
• W2023388639 cites W2097364162 @default.
• W2023388639 cites W2097498028 @default.
• W2023388639 cites W2102709391 @default.
• W2023388639 cites W2105459766 @default.
• W2023388639 cites W2114795136 @default.
• W2023388639 cites W2122099758 @default.
• W2023388639 cites W2125805909 @default.
• W2023388639 cites W2126912623 @default.
• W2023388639 cites W2134603462 @default.
• W2023388639 cites W2139681368 @default.
• W2023388639 cites W2142083551 @default.
• W2023388639 cites W2143815964 @default.
• W2023388639 cites W2144804038 @default.
• W2023388639 cites W2148433069 @default.
• W2023388639 cites W2148724380 @default.
• W2023388639 cites W2151929256 @default.
• W2023388639 cites W2151971588 @default.
• W2023388639 cites W2158538184 @default.
• W2023388639 cites W2160679445 @default.
• W2023388639 cites W3010233924 @default.
• W2023388639 cites W3025109026 @default.
• W2023388639 cites W4248208651 @default.
• W2023388639 doi "https://doi.org/10.1074/jbc.m700099200" @default.
• W2023388639 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17459883" @default.

• next