Studies of synthetic peptide analogs of the amphipathic helix. Effect of charge distribution, hydrophobicity, and secondary structure on lipid association and lecithin:cholesterol acyltransferase activation

Four peptides capable of forming an amphipathic alpha-helix have been synthesized and their conformational and lipid-binding properties studied. These peptides have been designed to vary the alpha-helix-forming potential as well as the charge distribution of the model peptide. The resulting peptide analogs and their complexes with dimyristoyl phosphatidylcholine were studied by using right angle light scattering, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, circular dichroism, intrinsic tryptophan fluorescence, and differential scanning calorimetry techniques. The four analogs, [Glu4,9, Leu11,17] (reverse-18A, [Glu4,9, Leu5,11,17] reverse-18A, [Glu1,8, Leu11,17] 18A, and [Glu1,8, Leu5,11,17] 18A were derived from a model amphipathic peptide Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) whose lipid-associating properties strongly mimic apolipoprotein A-I or derived from Lys-Trp-Leu-Asp-Ala-Phe-Tyr-Lys-Asp-Val-Ala-Lys-Glu-Leu-Glu-Lys-Ala-Phe (reverse-18A), a peptide with little affinity for lipid and having a reversed charge distribution compared to the 18A peptide. We have shown that by substituting glutamic acid and leucine for aspartic acid and alanine, respectively, in a weak lipid-associating amphipathic helix peptide, the lipid-associating ability can be increased. Thus, peptides with both kinds of charge distribution can associate with the lipid. The ability of the peptide to disrupt phospholipid bilayers, however, is higher for 18A analogs compared to the reverse-18A analogs even after increasing the helix-forming potential and hydrophobicity. In addition to forming smaller lipoprotein particles, the modified 18A analogs were much superior to the modified reverse-18A analogs in their ability to activate the enzyme lecithin:cholesterol acyltransferase. This demonstrates that the positions of charged residues in the amphipathic helix play an important role in lecithin:cholesterol acyltransferase activation.     

Studies of synthetic peptide analogs of the amphipathic helix. Structure of complexes with dimyristoyl phosphatidylcholine

The amphipathic helix hypothesis for the lipid-associating domains of exchangeable plasma apolipoproteins has been further studied by analysis of the structure of the complexes formed between four synthetic peptide analogs of the amphipathic helix and dimyristoyl phosphatidylcholine (DMPC). Density gradient ultracentrifugation, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, 1H NMR, high sensitivity differential scanning calorimetry, and circular dichroism were the techniques used in these studies. The two analogs Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) and 18A-Pro-18A whose sequences most strongly mimic native amphipathic sequences were found also most strongly to mimic apolipoprotein A-I in DMPC complex structure. The covalently linked dimer of the prototype amphipathic analog 18A, 18A-Pro-18A, appears to have greater lipid affinity than 18A. This presumably is the result of the cooperativity provided by two covalently linked lipid-associating domains in 18A-Pro-18A. The studies further suggest that the charge-reversed analog of the prototype 18A, reverse-18A, has the lowest lipid affinity of the four analogs studied and forms only marginally stable discoidal DMPC complexes. We postulate that this low lipid affinity is due predominantly, but not necessarily exclusively, to the lack of a hydrophobic contribution of lysine residues at the polar-nonpolar interface of reverse-18A versus 18A. The intermediate lipid affinity of des-Val10-18A, the fourth analog peptide, to produce a rank order of 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, supports this interpretation. Des-Val10-18A which has Val deleted from 18A has an amphipathic helical structure partially disrupted by the shift of 2 lysine residues away from the polar-nonpolar interface.     

Role of peptide structure in lipid-peptide interactions: nuclear magnetic resonance study of the interaction of pentagastrin and [Arg4]pentagastrin with dimyristoylphosphatidylcholine

Complexes formed between dimyristoylphosphatidylcholine (DMPC) and the peptide pentagastrin or [Arg4]pentagastrin were examined by 31P- and 2H-NMR. The cationic [Arg4]pentagastrin produces larger changes in the lipid NMR spectra than does the anionic pentagastrin. 31P-NMR spectra of DMPC with [Arg4]pentagastrin below the phase transition exhibits two components one of which is motionally restricted compared with the pure lipid. The exchange between these two lipid domains is slow on the millisecond time scale. The interactions between this peptide and phospholipid are diminished above the melting temperature of the complex. The 2H-NMR spectra of DMPC which had been labelled in a choline methylene group is also affected more by the [Arg4]pentagastrin than by pentagastrin. In the presence of [Arg4]pentagastrin, even above the lipid phase transition, an additional doublet with a smaller quadrupole splitting is observed. These results clearly demonstrate the importance of peptide charge in determining the effects of peptides on lipid bilayers.     

Effect of leucine to phenylalanine substitution on the nonpolar face of a class A amphipathic helical peptide on its interaction with lipid: high resolution solution NMR studies of 4F-dimyristoylphosphatidylcholine discoidal complex

Model class A amphipathic helical peptides mimic several properties of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins. Previously, we reported the NMR structures of Ac-18A-NH(2) (renamed as 2F because of two phenylalanines), the base-line model class A amphipathic helical peptide in the presence of lipid ( Mishra, V. K., Anantharamaiah, G. M., Segrest, J. P., Palgunachari, M. N., Chaddha, M., Simon Sham, S. W., and Krishna, N. R. (2006) J Biol. Chem. 281, 6511-6519 ). Substitution of two Leu residues on the nonpolar face (Leu(3) and Leu(14)) with Phe residues produced the peptide 4F (so named because of four phenylalanines), which has been extensively studied for its anti-inflammatory and antiatherogenic properties. Like 2F, 4F also forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Since subtle structural changes in the peptide-lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of 4F in 4F.DMPC discs. Like 2F, 4F adopts a well defined amphipathic alpha-helical structure in association with the lipid at a 1:1 peptide/lipid weight ratio. Nuclear Overhauser effect (NOE) spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. Similar to 2F, the pattern of observed peptide-lipid NOEs is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). However, in contrast to 2F in 2F.DMPC, 4F in the 4F.DMPC complex is located closer to the lipid headgroup as evidenced by a number of NOEs between 4F and DMPC headgroup protons. These NOEs are absent in the 2F.DMPC complex. In addition, the conformation of the DMPC sn-3 chain in 4F.DMPC complex is different than in the 2F.DMPC complex as evidenced by the NOE between lipid 2.CH and betaCH(2) protons in 4F.DMPC, but not in 2F.DMPC, complex. Based on the results of this study, we infer that the antiatherogenic properties of 4F may result from its preferential interaction with lipid headgroups.     

The structure around the thioester bond in bovine alpha 2-macroglobulin. Possible implications for the conformational stability of the inhibitor on thioester cleavage

The residues contributing to the thioester bonds in bovine alpha 2-macroglobulin were differentially labelled by modification of the Glu moiety with [14C]methylamine and of the Cys moiety with iodo[3H]acetate. The labelled region was identified and analyzed in a tryptic peptide. Two amino acid replacements between human and bovine alpha 2-macroglobulin were found at positions +3 (Val/Ala) and +4 (Leu/Arg) from the Glu moiety of the thioester. Thus, marked differences exist between the human and bovine proteins in side chain size and charge close to the thioester bonds. These differences may explain the greater conformational stability of bovine alpha 2-macroglobulin, compared with that of the human inhibitor, after cleavage of the thioester bonds.     

Lipid bilayer perturbations induced by simple hydrophobic peptides

Mixtures of tripeptides of the form Ala-X-Ala-O-tert-butyl with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers have been used as a model system for studying the influence of hydrophobic peptides on membrane order and dynamic properties by means of deuterium NMR spectroscopy. Tripeptides with X = Ala, Leu, Phe, and Trp have been examined. Lipid 2H NMR spectra of acyl chain perdeuteriated DMPC ([2H54]DMPC) show that the addition of peptide disorders the bilayer lipid acyl chains and that the extent of the perturbation increases as the size of the central residue increases. Moment analyses of the spectra indicate that, while the average acyl chain order parameter decreases with increasing central residue size, the order parameter spread across the bilayer (the mean-squared width of the distribution) increases. Lipid segmental 2H longitudinal relaxation rates, 1/T1(i), exhibit a square-law functional dependence on SCD(i) both with and without the addition of peptide. The addition of peptide causes an increase in the slope of plots of 1/T1(i) vs. (SCD(i))2 with little change in the 1/T1(i) intercept, indicating a complex modulation of the acyl chain motions. 2H NMR spectra of Ala-[2H4]Ala-Ala-O-tert-butyl in DMPC bilayers have both isotropic and powder pattern components that vary as a function of temperature. At 30 degrees C the 2H spin-lattice relaxation times for the labeled Ala residue increase in going from bilayer-incorporated peptide to polycrystalline peptide to polycrystalline Ala.HCl. These experiments provide no information on the location of these peptides in the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solid‐state NMR and simulation studies of equinatoxin II N‐terminus interaction with lipid bilayers

The interaction with model membranes of a peptide, EqtII_1–32, corresponding to the N‐terminal region of the pore‐forming toxin equinatoxin II (EqtII) has been studied using solid‐state NMR and molecular dynamics (MD) simulations. The distances between specifically labeled nuclei in [¹⁹F‐para]Phe16‐[1‐¹³C]Leu19 and [¹⁹F‐para]Phe16‐[¹⁵N]Leu23 analogs of EqtII_1–32 measured by REDOR in lyophilized peptide were in agreement with published crystal and solution structures. However, in both DMPC and mixed DMPC:SM membrane environments, significant changes in the distances between the labeled amino acid pairs were observed, suggesting changes in helical content around the experimentally studied region, 16–23, in the presence of bilayers. ¹⁹F‐³¹P REDOR experiments indicated that the aromatic ring of Phe16 is in contact with lipid headgroups in both membrane environments. For the DMPC:SM mixed bilayers, a closer interaction between Phe16 side chains and lipid headgroups was observed, but an increase in distances was observed for both labeled amino acid pairs compared with those measured for EqtII_1–32 in pure DMPC bilayers. The observed differences between DMPC and DMPC:SM bilayers may be due to the greater affinity of EqtII for the latter. MD simulations of EqtII_1–32 in water, on a pure DMPC bilayer, and on a mixed DMPC:SM bilayer indicate significant peptide secondary structural differences in the different environments, with the DMPC‐bound peptide adopting helical formations at residues 16–24, whereas the DMPC:SM‐bound peptide exhibits a longer helical stretch, which may contribute to its enhanced activity against PC:SM compared with pure PC bilayers. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Nuclear magnetic resonance investigation of the interactions with phospholipid of an amphipathic alpha-helix-forming peptide of the apolipoprotein class

To further understand the packing of amphipathic alpha-helices of apolipoproteins in serum lipoproteins, we have investigated the interactions with dimyristoyl phosphatidylcholine (DMPC) of a 13C-labeled, 18-residue peptide (18A) which can form an amphipathic alpha-helix. This peptide whose amino acid sequence is DWLKAFYDKVAEKLKEAF has the positive-negative residue clustering typical of the apolipoprotein class of amphipathic helix. 13CH3-alanine was introduced as the 11th residue of 18A so that the 13CH3 group protrudes on the apolar side of the amphipathic helix. [13C]NMR spectra of [13C-Ala11]18A in discoidal complexes with DMPC show three resonances from the Ala-13CH3 group; one originates from 18A in aqueous solution, while those at chemical shifts (delta) of 15.2 and 16.4 ppm are assigned to 18A in the "edge" and "faces," respectively, of the discoidal complex. The proportion of 18A in the faces of the discoidal complex increases as the size of the disk is increased by raising the lipid/peptide ratio. 18A covers the edge of the disk so that the 13CH3-Ala side chain from these molecules is in contact with DMPC acyl chains. [13C-Ala11]18A bound to the surface of an egg PC small unilamellar vesicle gives a single resonance from 18A at delta 16.3 ppm consistent with there being no edge location. Cooling 18A-DMPC disks to 15 degrees C crystallizes the DMPC bilayer and restricts the motion of the 13CH3-Ala group of the 18A molecules. The molecular motions of the side chains of the amphipathic helix are sensitive to their location in the disk and to PC molecular packing.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

Amino acid sequences of two tryptic peptides from D(-)-beta-hydroxybutyrate dehydrogenase radiolabeled at essential carboxyl and sulfhydryl groups

D(-)beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains essential thiol and carboxyl groups. A tryptic BDH peptide labeled at an essential thiol with [3H]N-ethylmaleimide (NEM), and another tryptic peptide labeled at an essential carboxyl with N,N'-dicyclohexyl [14C]carbodiimide (DCCD), were isolated and sequenced. The peptide labeled with [3H]NEM had the sequence Met.Glu.Ser.Tyr.Cys*.Thr.Ser. Gly.Ser.Thr.Asp.Thr.Ser.Pro.Val.Ile.Lys. The label was at Cys. The same peptide was isolated from tryptic digests of BDH labeled at its nucleotide-binding site with the photoaffinity labeling reagent, arylazido- -[3-3H] alanyl-NAD. These results suggest that the essential thiol of BDH is located at its nucleotide-binding site, and agree with our previous observation that NAD and NADH protect BDH against inhibition by thiol modifiers. The [14C]DCCD-labeled peptide had the sequence Glu.Val.Ala.Glu*.Val. Asn. Leu.Trp.Gly.Thr.Val.Arg. DCCD appeared to modify the glutamic acid residue marked by an asterisk. Sequence analogies between these peptides and other proteins have been discussed.     

Assignment of the 13C-NMR spectra of virgin and reactive-site modified turkey ovomucoid third domain

The virgin (reactive-site Leu18-Glu19 peptide bond intact) and modified (reactive-site Leu18-Glu19 peptide bond hydrolyzed) forms of turkey ovomucoid third domain (OMTKY3 and OMTKY3*, respectively) have been analyzed by proton-detected 1H(13C) two-dimensional single-bond correlation (1H[13C]SBC) spectroscopy. Previous 1H-nmr assignments of these proteins [A.D. Robertson, W.M. Westler, and J.L Markley (1988) Biochemistry, 27, 2519-2529; G. I. Rhyu and J. L. Markley (1988) Biochemistry, 27, 2529-2539] have been extended to directly bonded 13C atoms. Assignments have been made to 52 of the 56 backbone 13C alpha-1H units and numerous side-chain 13C-1H groups in both OMTKY3 and OMTKY3*. The largest changes in the 13C chemical shift upon conversion of OMTKY3 to OMTKY3* occur at or near the reactive site, and tend toward values observed in small peptides. Moreover, the side-chain prochiral methylene protons attached to the C gamma of Glu19 and C delta of Arg21 show nonequivalent chemical shifts in OMTKY3 but more equivalent chemical shifts in OMTKY3*. These results suggest that the reactive site region becomes less ordered upon hydrolysis of the Leu18-Glu19 peptide bond. Comparison of 13C alpha chemical shifts of OMTKY3 and bovine pancreatic trypsin inhibitor [D. Brühuiler and G. Wagner (1986) Biochemistry 25, 5839-5843; N. R. Nirmala and G. Wagner (1988) Journal of the American Chemical Society, 110, 7557-7558] with small peptide values [R. Richarz and K. Wüthrich (1978) Biopolymers, 17, 2133-2141] suggests that 13C alpha chemical shifts of residues residing in helices are generally about 2 ppm downfield of resonances from nonhelical residues.     

Toward understanding the role of insulin alpha-helix II in the growth-promoting activity of insulin.

For further understanding the contribution of the alpha-helix II (alphaII) in the growth-promoting activity of insulin, the residues A2Ile, A5Gln, and A8Thr located in alphaII were mutated to Leu, Glu, and Tyr, respectively. Three mutant insulins, [A2Leu]human insulin, [A5Glu]human insulin, and [A8Tyr]human insulin, were prepared by means of site-directed mutagenesis. The in vitro growth-promoting activities of the three mutant insulins, measured using GR2H6 cells, were 7.5%, 291%, and 250% of that of native insulin, respectively. Their receptor-binding activities to the insulin receptor were 2.3%, 46.7%, and 138.7%, respectively, compared with native insulin. Both the growth-promoting and receptor-binding activities of [A2Leu]human insulin and [A3Leu]insulin (Shi et al., 1997) were parallel and greatly decreased compared with native insulin. The results demonstrate that the residues A2Ile and A3Val in the alphaII are essential for the growth-promoting activity of insulin, and the growth-promoting function of insulin might be performed through, or mainly through, binding to the insulin receptor. The growth-promoting activities of [A5Glu]human insulin and [A8Tyr]human insulin were increased 6-fold and 2-fold, respectively, compared with native insulin, indicating that their growth-promoting activities might be expressed by, or mainly by, binding to the IGF-1 receptor.     

Pyruvate carboxylase: isolation of the biotin-containing tryptic peptide and the determination of its primary sequency

The biotin-containing tryptic peptides of pyruvate carboxylase from sheep, chicken, and turkey liver mitochondria have been isolated and their primary structures determined. The amino acid sequences of the 19 residue peptides from chicken and turkey are identical and share a common sequence of 14 residues around biocytin with the 24-residue peptide isolated from sheep. The sequences obtained were: residue 1 → 11 Avian: Gly Ala Pro Leu Val Leu Ser Ala Met Biocytin Met Sheep: Gly Gln Pro Leu Val Leu Ser Ala Met Biocytin Met residues 12 → 19 or 24 Avian: Glu Thr Val Val Thr Ala Pro Arg Sheep: Glu Thr Val Val Thr Ser Pro Val Thr Glu Gly Val Arg A sensitive radiochemical assay for biotin was developed based on the tight binding of biotin by avidin. The ability of zinc sulfate to precipitate, without dissociating, the avidin-biotin complex provided a convenient procedure for separating free and bound biotin, and hence, for back-titrating a standard amount of avidin with [¹⁴C]biotin.     

Conformation and lipid binding of the N-terminal (1-44) domain of human apolipoprotein A-I

Because of its role in reverse cholesterol transport, human apolipoprotein A-I is the most widely studied exchangeable apolipoprotein. Residues 1-43 of human apoA-I, encoded by exon 3 of the gene, are highly conserved and less well understood than residues 44-243, encoded by exon 4. In contrast to residues 44-243, residues 1-43 do not contain the 22 amino acid tandem repeats thought to form lipid binding amphipathic helices. To understand the structural and functional roles of the N-terminal region, we studied a synthetic peptide representing the first 44 residues of human apoA-I ([1-44]apoA-I). Far-ultraviolet circular dichroism spectra showed that [1-44]apoA-I is unfolded in aqueous solution. However, in the presence of n-octyl beta-d-glucopyranoside, a nonionic lipid mimicking detergent, above its critical micelle concentration ( approximately 0.7% at 25 degrees C), sodium dodecyl sulfate, an ionic detergent, above its CMC ( approximately 0.2%), trimethylamine N-oxide, a folding inducing organic osmolyte, or trifluoroethanol, an alpha-helix inducer, alpha-helical structure was formed in [1-44]apoA-I up to approximately 45%. Characterization by density gradient ultracentrifugation and visualization by negative staining electron microscopy demonstrated that [1-44]apoA-I interacts with dimyristoylphosphatidylcholine (DMPC) over a wide range of lipid:peptide ratios from 1:1 to 12:1 (w/w). At 1:1 DMPC:[1-44]apoA-I (w/w) ratio, discoidal complexes with composition approximately 4:1 (w/w) and approximately 100 A diameter were formed in equilibrium with free peptide. At higher ratios, discoidal complexes were shown to exist together with a heterogeneous population of lipid vesicles with peptide bound also in equilibrium with free peptide. When bound to DMPC, [1-44]apoA-I has approximately 60% helical structure, independent of whether it forms discoidal or vesicular complexes. This helical content is consistent with that of the predicted G helix (residues 8-33). Our data provide the first strong and direct evidence that the N-terminal region of apoA-I binds lipid and can form discoidal structures and a heterogeneous population of vesicles. In doing so, approximately 60% of this region folds into alpha-helix from random coil. The composition of the 100 A discoidal complex is approximately 5 [1-44]apoA-I and approximately 150 DMPC molecules per disk. The helix length of 5 [1-44]apoA-I molecules in lipid-bound form is just long enough to wrap around the DMPC bilayer disk once.     

A comparison of the interaction of glucagon, human parathyroid hormone-(1-34)-peptide and calcitonin with dimyristoylphosphatidylglycerol and with dimyristoylphosphatidylcholine

The interaction of glucagon, human parathyroid hormone-(1-34)-peptide and salmon calcitonin with dimyristoylphosphatidylglycerol (DMPG) and with dimyristoylphosphatidylcholine (DMPC) was studied as a function of pH and temperature. The effect of lipid on the secondary structure of the peptide was assessed by circular dichroism and the effect of the peptide on the phase transition properties of the lipid was studied using differential scanning calorimetry. Some peptides interact more strongly with anionic than with zwitterionic phospholipids. This does not require an overall positive charge on the peptide. Increased thermal stability is observed in complexes formed between cationic peptides and anionic lipids. Particularly marked effects of glucagon and human parathyroid hormone-(1-34)-peptide on the phase transition properties of DMPG at pH 5 have been observed. The transition temperature is raised over 10 degrees C at a lipid/peptide molar ratio of less than 30:1 and the transition enthalpy is increased over 2-fold. These effects do not occur with any basic peptide and were not observed with metorphinamide, molluscan cardioexcitatory neuropeptide or myelin basic protein. The results demonstrate that certain peptides can affect the phase transition properties of lipids in a manner similar to divalent cations. The overall hydrophobicities of these peptides can be evaluated by their partitioning between aqueous and organic solvents. None of the above three peptide hormones partition into the organic phase. However, a closely related peptide, human calcitonin, does exhibit substantial partitioning into the organic phase. Nevertheless, human calcitonin has a weaker interaction with both DMPC and DMPG than does salmon calcitonin. The effects of human calcitonin on the phase transition of DMPC are qualitatively different from those of salmon calcitonin in that the human form more readily eliminates the pretransition but causes less change in the main transition. Like overall charge, overall hydrophobicity is not an overwhelming factor in determining the ability of peptides to interact with phospholipids but rather more specific interactions are required for strong complexes to form.     

Study of the effect of poly(L-lysine) on phosphatidic acid and phosphatidylcholine/phosphatidic acid bilayers by raman spectroscopy

The effect of polylysine (PLL) on dimyristoylphosphatidic acid (DMPA), on dimyristoyl-phosphatidylcholine (DMPC), and on mixtures of these lipids was investigated by Raman spectroscopy. These results show that long polylysine (Mr approximately 200,000) increases the stability of the acyl chain matrix of DMPA to form a more closely packed structure with a stoichiometry of one lysine residue per PA molecule. On the other hand, short PLL (Mr 4000) destabilizes the PA bilayer, and the complex formed undergoes a gel to liquid-crystalline transition at a lower temperature than of the pure lipid. For both cases, we have observed that bound polylysine adopts a beta-sheet conformation as opposed to the alpha-helical structure previously found for dipalmitoylphosphatidylglycerol/long PLL complexes [Carrier, D., & Pézolet, M. (1984) Biophys. J. 46, 497-506]. The difference in the thermal behavior of complexes of DMPA with long and short polylysines is believed to be associated with the fact that in the complex the long polypeptide adopts the beta-sheet conformation over the whole range of temperatures investigated while the short one undergoes a change of conformation from beta-sheet of random coil upon heating. Therefore, the conformation of the lipid-bound polypeptides depends on the nature of the polar head group of the lipid, not only on its net charge, and it affects considerably the thermotropism of the lipid. On the other hand, both long and short polylysines show no affinity for phosphatidylcholine since the temperature profiles of DMPC and of DMPC/PLL complexes exhibit exactly the same behavior.(ABSTRACT TRUNCATED AT 250 WORDS)     

ZK98299--a new antiprogesterone: biochemical characterization of steroid binding parameters in the calf uterine cytosol.

We have examined steroid binding characteristics of a newly synthesized antisteroid, ZK98299 [onapristone, 11 beta-(4-dimethylaminophenyl)-17 alpha-hydroxy-17 beta-(3-hydroxypropyl)- 13 alpha-methyl-4,9-gonadien-3-one], in the calf uterus cytosol and compared the nature of this interaction with the binding of progesterone receptor (PR) agonist R5020 [promegestone, 17,21-dimethylpregna-4,9-diene-3,20-dione]. In the freshly prepared cytosol, [3H]ZK98299 interacted specifically with a macromolecule: the binding was abolished in the presence of excess progestins (R5020 and progesterone) and the antiprogesterone ZK98299. The high affinity (Kd = 2.5 nM) interaction between [3H]ZK98299 and PR was temperature- and time-dependent, reaching an optimum by 2-3 h at 0 degrees C, and was facilitated by 20 mM Na2MoO4. Under nontransforming conditions, [3H]ZK98299-receptor complexes sedimented as 8 S species in 8-30% linear glycerol gradients. Upon salt or thermal transformation, there was a loss of the 8 S form, with only a small fraction of total complexes (5-7%) binding to DNA-cellulose. In contrast, transformed [3H]R5020-receptor complexes exhibited a greater extent of binding (25-55%) to DNA-cellulose. [3H]ZK98299-receptor complexes could be resolved into two ionic species over DEAE-Sephacel following incubation of the complexes at 0 or 23 degrees C. [3H]ZK98299 binding was sensitive to sulfhydryl group modification as beta-mercaptoethanol increased the extent of steroid binding. Although treatment with iodoacetamide (IA) abolished [3H]R5020 binding, there was a significant (nearly twofold) increase in the [3H]ZK98299 binding. The results of this study point to similarities and differences between the steroid binding properties of the uterine PR occupied by R5020 and ZK98299: both steroids appear to bind the same 8 S receptor but exhibit differential DNA binding and sensitivity to IA. The reported antagonist properties of ZK98299 may, therefore, be explained on the basis of a distinct receptor conformation induced by the antisteroid.     

The essential role of specific Halobacterium halobium polar lipids in 2D-array formation of bacteriorhodopsin.

The mechanism whereby bacteriorhodopsin (BR), the light driven proton pump from the purple membrane of Halobacterium halobium, arranges in a 2D-hexagonal array, has been studied in bilayers containing the protein, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and various fractions of H. halobium membrane lipids, by freeze fracture electron microscopy and examination of optical diffractograms of the micrographs obtained. Electron micrographs of BR/DMPC complexes containing the entire polar lipid component of H. halobium cell membranes or the total lipid component of the purple membrane, with a protein-to-total lipid molar ratio of less than 1:50 and to which 4 M NaCl had been added, revealed that trimers of BR formed into an hexagonal 2D-array similar to that found in the native purple membrane, suggesting that one or more types of the purple membrane polar lipids are required for array formation. To support this suggestion, bacteriorhodopsin was purified free of endogenous purple membrane lipids and reconstituted into lipid bilayer complexes by detergent dialysis. The lipids used to form these complexes are 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC) as the major lipid and, separately, each of the individual lipid types from the H. halobium cell membranes, namely 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-phosphate (DPhPGP), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-sulphate (DPhPGS), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol (DPhPG) and 2,3-di-O-phytanyl-1-O-[beta-D-Galp-3-sulphate-(1----6)-alpha-D- Manp-(1----2)-alpha-D-Glcp]-sn-glycerol (DPhGLS). When examined by freeze-fracture electron microscopy, only the complexes containing 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol- 1'-phosphate or 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol-1'-sulphate, at high protein density (less than 1:50, bacteriorhodopsin/phospholipid, molar ratio) and to which 4 M NaCl had been added, showed well defined 2D hexagonal arrays of bacteriorhodopsin trimers similar to those observed in the purple membrane of H. halobium.     

The membrane interaction of amphiphilic model peptides affects phosphatidylserine headgroup and acyl chain order and dynamics. Application of the "phospholipid headgroup electrometer" concept to phosphatidylserine

Deuterium nuclear magnetic resonance (2H NMR) was used to study the interaction of amphiphilic model peptides with model membranes consisting of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine deuterated either at the beta-position of the serine moiety ([2-2H]DOPS) or at the 11-position of the acyl chains ([11,11-2H2]DOPS). The peptides are derived from the sequences H-Ala-Met-Leu-Trp-Ala-OH (AX, one-letter code with X = MLWA) and H-Arg-Met-Leu-Trp-Ala-OH (RX+) and contain a positive charge of +1 (AXme+) or +2 (RXme2+) at the amino terminus or one positive charge at each end of the molecule (AXetN2+). Upon titration of dispersions of DOPS with the peptides, the divalent peptides show a similar extent of binding to the DOPS bilayers, which is larger than that of the single charged peptide. Under these conditions the values of the quadrupolar splitting (delta vq) of both [2-2H]DOPS and [11,11-2H2]DOPS are decreased, indicating that the peptides reduce the order of both the DOPS headgroup and the acyl chains. The extent of the decrease depends on the amount of peptide bound and on the position of the charged moieties in the peptide molecule. The effects exerted by the peptides on the delta vq value of [2-2H]DOPS are consistent with the PS headgroup responding as a molecular electrometer to the surface charge resulting from the presence of the peptides in the lipid-water interface. The effects on the acyl chain deuterons are in agreement with a localization of the peptides intercalated in between the lipid headgrouops.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of DMPC- and DLPE-based lipid bilayers.

A 250 ps molecular dynamics simulation of the dimyristoylphosphatidylcholine (DMPC)-based lipid bilayer, including explicit water molecules, is reported. The solvent environment of the head groups and other structural properties of the bilayer have been analyzed and compared with experimental results as well as our previous simulation of the dilauroylphosphatidylethanolamine (DLPE)-based bilayer. From this comparison we find that the solvent structure around the DMPC head group (clathrate shell) is significantly different than that around the DLPE head group (typical hydrogen bonding interactions). We have modeled the probable relationship between the different solvent environments around the R-N(CH3)3+ (DMPC) and R-NH3+ (DLPE) head groups and the different interlammelar distances in these systems by performing potential of mean force (PMF) simulations on two N(CH3)4+ and NH4+ ions in water. From the PMF simulations it appears that the differences in the hydration of the DMPC and DLPE head groups is not responsible for the differences in the hydration force observed for these systems. We also find that the orientational polarization of DLPE and DMPC is similar, which suggests that solvent polarization is not responsible for the differences in the hydration repulsion behavior observed in these systems. We also examined the order parameters for DMPC and found them to be in reasonable agreement with experiment. Given the different characteristics of the DLPE and DMPC head groups, we suggest an explanation of the differences in the interlammellar spacings of bilayers composed of these like-charged lipids. From our DLPE simulations we find that the R-NH3+ head groups can interact with the nonesterified oxygens of the phosphate group in an intraleaflet or an interleaflet manner. For the latter a "cross link" between two leaflets can be formed, which causes a stabilization of the interlamellar spacings at fairly short distances. Moreover, due to the strong intraleaflet interaction we find that the DLPE interface is relatively "flat" (as opposed to DMPC-based bilayers), which results in a surface that has regions of positive and negative charge that reside in the same plane along the bilayer normal. Based on this we propose that the DLPE bilayer interface can correlate itself with another DLPE interface by alignment of the regions of positive (or negative) charge on one leaflet with the opposite charges on the opposing leaflet.