Recognition of alpha-helical peptide structures using high-performance liquid chromatographic retention data for D-amino acid analogues: influence of peptide amphipathicity and of stationary phase hydrophobicity

The reversed-phase HPLC behaviour of double D-amino acid replacement sets of amphipathic and non-amphipathic helix-forming peptides consisting exclusively of leucine, lysine and alanine residues was studied on different polymer-encapsulated silica-based stationary phases. Plotting the retention times versus the position of D-amino acid substitution gives a characteristic pattern showing decreased retention times in the helical region. The retention time profile obtained using an amphipathic alpha-helix is caused by disturbance of the preferred binding domain of the stationary phase-bound peptide. However, the effect is similar but less pronounced using a non-amphipathic helical peptide that is unable to interact by a preferred binding site. The results demonstrate that reversed-phase HPLC data for peptide analogues provide an indication event of a non-amphipathic helical structure in peptides.     

Simulation of voltage-dependent interactions of alpha-helical peptides with lipid bilayers

Pore formation in lipid bilayers by channel-forming peptides and toxins is thought to follow voltage-dependent insertion of amphipathic alpha-helices into lipid bilayers. We have developed an approximate potential for use within the CHARMm molecular mechanics program which enables one to simulate voltage-dependent interaction of such helices with a lipid bilayer. Two classes of helical peptides which interact with lipid bilayers have been studied: (a) delta-toxin, a 26 residue channel-forming peptide from Staphylococcus aureus; and (b) synthetic peptides corresponding to the alpha 5 and alpha 7 helices of the pore-forming domain of Bacillus thuringiensis CryIIIA delta-endotoxin. Analysis of delta-toxin molecular dynamics (MD) simulations suggested that the presence of a transbilayer voltage stabilized the inserted location of delta-toxin helices, but did not cause insertion per se. A series of simulations for the alpha 5 and alpha 7 peptides revealed dynamic switching of the alpha 5 helix between a membrane-associated and a membrane-inserted state in response to a transbilayer voltage. In contrast the alpha 7 helix did not exhibit such switching but instead retained a membrane associated state. These results are in agreement with recent experimental studies of the interactions of synthetic alpha 5 and alpha 7 peptides with lipid bilayers.     

The amphipathic helix concept: length effects on ideally amphipathic LiKj(i=2j) peptides to acquire optimal hemolytic activity

In a minimalist approach to modeling lytic toxins, amphipathic peptides of LiKj with i=2j composition and whose length varies from 5 to 22 residues were studied for their ability to induce hemolysis and lipid vesicle leakage. Their sequences were designed to generate ideally amphipathic alpha helices with a single K residue per putative turn. All the peptides were lytic, their activities varying by more than a factor of 103 from the shortest 5-residue-long peptide (5-mer) to the longest 22-mer. However, there was no monotonous increase versus length. The 15-mer was as active as the 22-mer and even more than melittin which is used as standard. Partition coefficients from the buffer to the membrane increased in relation to length up to 12 residues, then weakly decreased to reach a plateau, while they were expected to increase monotonously with peptide length and hydrophobicity as revealed from HPLC retention times. Fluorescence labeling by a dansyl group at the N-terminus, or by a W near the CO-terminus, show that up to 12 residues, the peptides were essentially monomeric while longer peptides strongly aggregated in the solution. Lipid affinity was then controlled by peptide length and was found to be limited by folding and self-association in buffer. The lytic activity resulted both from lipid affinity, which varied by a factor of 20-fold, and from efficiency in disturbing the membrane when bound, the latter steeply and monotonously increasing with length. The 15-residue-long peptide, KLLKLLLKLLLKLLK, had the optimal size for highest lytic activity. The shallow location of the fluorescent labels in the lipids is further evidence for a model of peptides remaining flat at the interface.     

Von Recklinghausen''s disease (neurofibromatosis) and pregnancy: apropos of a clinical case

Using a random combinatorial synthetic peptide library method based on a one-bead one-peptide concept for ligand identification (Lam et. al, Nature 1991, 354, 82-84.), idiotype specific peptides were retrieved and optimized for interaction with the cell surface immunoglobulins [IgM(kappa)] of two murine B lymphoma cell lines. Several of the identified peptides were characterized with respect to cell binding and signal transduction. These peptides were able to bind specifically to the surface immunoglobulins of these lymphoma cells. In addition to binding, when synthesized in tetrameric or multimeric forms, the peptides were able to trigger signal transduction resulting in an increase in protein tyrosine phosphorylation. Since D-amino acid peptide libraries were used in some of our efforts to identify binding ligands, several of the idiotype-specific peptides are composed of all D-amino acids (e.g. wGeyvmvnG). These findings may have important therapeutic implications for targeted-therapy of B-cell lymphoma as these D-amino acid ligands are more resistant to proteolysis resulting in a prolonged pharmacokinetic disposition in vivo.     

Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics

Analysis of antimicrobial activities that are present in the skin secretions of the South American frog Phyllomedusa bicolor revealed six polycationic (lysine-rich) and amphipathic alpha-helical peptides, 24-33 residues long, termed dermaseptins B1 to B6, respectively. Prepro-dermaseptins B all contain an almost identical signal peptide, which is followed by a conserved acidic propiece, a processing signal Lys-Arg, and a dermaseptin progenitor sequence. The 22-residue signal peptide plus the first 3 residues of the acidic propiece are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The 25-residue amino-terminal region of prepro-dermaseptins B shares 50% identity with the corresponding region of precursors for D-amino acid containing opioid peptides or for antimicrobial peptides originating from the skin of distantly related frog species. The remarkable similarity found between prepro-proteins that encode end products with strikingly different sequences, conformations, biological activities and modes of action suggests that the corresponding genes have evolved through dissemination of a conserved "secretory cassette" exon.     

Microscopic observations of the different morphological changes caused by anti-bacterial peptides on Klebsiella pneumoniae and HL-60 leukemia cells

Natural anti-bacterial peptides cecropin B (CB) and its analogs cecropin B-1 (CB-1), cecropin B-2 (CB-2) and cecropin B-3 (CB-3) were prepared. The different characteristics of these peptides, with amphipathic/hydrophobic alpha-helices for CB, amphipathic/amphipathic alpha-helices for CB-1/CB-2, and hydrophobic/hydrophobic alpha-helices for CB-3, were used to study the morphological changes in the bacterial cell, Klebsiella pneumoniae and the leukemia cancer cell, HL-60, by scanning and transmission electron microscopies. The natural and analog peptides have comparable secondary structures as shown by circular dichroism measurements. This indicates that the potency of the peptides on cell membranes is dependent of the helical characteristics rather than the helical strength. The microscopic results show that the morphological changes of the cells treated with CB are distinguishably different from those treated with CB-1/CB-2, which are designed to have enhanced anti-cancer properties by having an extra amphipathic alpha-helix. The morphological differences may be due to their different modes of action on the cell membranes resulting in the different potencies with lower lethal concentration and higher concentration of 50% inhibition (IC50) of CB on bacterium and cancer cell, respectively, as compared with CB-1/:CB-2 (Chen et al. 1997. Biochim. Biophys. Acta 1336, 171-179). In contrast, CB-3 has little effect on either the bacterium or the cancer cell. These results provide microscopic evidence that different killing pathways are involved with the peptides.     

Structure-function relationships in side chain lactam cross-linked peptide models of a conserved N-terminal domain of apolipoprotein E

Bioactive peptides have multiple conformations in solution but adopt well-defined conformations at lipid surfaces and in interactions with receptors. We have used side chain lactam cross-links to stabilize secondary structures in the following peptide models of a conserved N-terminal domain of apolipoprotein E (cross-link periodicity in parentheses): I, H2N-GQTLSEQVQEELLSSQVTQELRAG-COOH (none); III, [sequence; see text] (i to i + 3); IV,[sequence; see text] (i to i + 4); IVa, [sequence, see text] (i to i + 4) (lactams above the sequence, potential salt bridges below the sequence). We previously demonstrated [Luo et al. (1994) Biochemistry 33, 12367-12377; Braddock et al. (1996) Biochemistry 35, 13975-13984] that peptide III, containing lactam cross-links between the i and i + 3 side chains, enhances specific binding of LDL via a receptor other than the LDL-receptor. Peptide III in solution consists of two short alpha helices connected by a non alpha helical segment. Here we examine the hypothesis that the domain modeled by peptide III is one antipode of a conformational switch. To model another antipode of the switch, we introduced two strategic modifications into peptide III to examine structure-function relationships in this domain: (1) the spacing of the lactam cross-links was changed (i to i + 4 in peptides IV and IVa) and (2) peptides IV and IVa contain the two alternative sequences at a site of a possible end-capping interaction in peptide III. The structure of peptide IV, determined by 2D-NMR, is alpha helical across its entire length. Despite the remarkable degree of structural order, peptide IV is biologically inactive. In contrast, peptides III and possibly IVa contain a central interruption of the alpha helix, which appears necessary for biological activity. These and other studies support the hypothesis that this domain is a conformational switch which, to the extent that it models apolipoprotein E itself, may modulate interactions between apo E and its various receptors.     

Absorption and excretion of undegradable peptides: role of lipid solubility and net charge

Absorption and excretion of undegradable peptides were investigated with use of octapeptides synthesized from D-amino acids. D-Tyrosine was included in each peptide to permit labeling with 125I, D-glutamic acid or D-lysine were included to vary net electric charge and D-serine or D-leucine were included to vary lipid solubility. Peptides were administered parenterally or orally to normal rats drinking 5% glucose or maltose. Forty-five percent of a lipid-insoluble, negatively charged octapeptide added to the drinking fluid in milligram quantities was absorbed from the intestine and excreted intact in urine; 90% of this peptide was recovered in urine after parenteral injection. In contrast, lipophilic D-octapeptides were largely excreted in feces, even after subcutaneous injection; the amounts excreted in feces were correlated with oil/aqueous partition coefficients. Evidence is presented that lipophilic peptides entering liver cells combine with bile salts to form hydrophilic complexes that are secreted rapidly at high concentration in bile. At physiological concentrations of bile salts (5-40 mM) and nanomolar concentrations of peptide the binding is so complete that these undegradable peptides are rapidly cleared from liver to duodenal fluid in association with the bile salts. After reaching the ileum the bile salts are reabsorbed to blood, leaving the original lipophilic peptides to be excreted in the feces from which they can be extracted, purified and identified by high-pressure liquid chromatography. These mechanisms are discussed in relation to a) the paracellular absorption of peptides and other solutes by solvent drag and b) the delivery and fate of biologically active peptides.     

Structure-function relationship of antibacterial synthetic peptides homologous to a helical surface region on human lactoferrin against Escherichia coli serotype O111

Lactoferricin includes an 11-amino-acid amphipathic alpha-helical region which is exhibited on the outer surface of the amino-terminal lobe of lactoferrin. Synthetic peptides homologous to this region exhibited potent antibacterial activity against a selected range of both gram-negative and gram-positive bacteria. An analog synthesized with methionine substituted for proline at position 26, which is predicted to disrupt the helical region, abolished antibacterial activity against Escherichia coli and considerably reduced antibacterial activity against Staphylococcus aureus and an Acinetobacter strain. The mode of action of human lactoferrin peptide (HLP) 2 against E. coli serotype O111 (NCTC 8007) was established by using flow cytometry, surface plasmon resonance, and transmission electron microscopy. Flow cytometry was used to monitor membrane potential, membrane integrity, and metabolic processes by using the fluorescent probes bis-1,3-(dibutylbarbituric acid)-trimethine oxonol, propidium iodide, and carbonyl cyanide m-chlorophenylhydrazone, respectively. HLP 2 was found to act at the cell membrane, causing complete loss of membrane potential after 10 min and of membrane integrity within 30 min, with irreversible damage to the cell as shown by rapid loss of viability. The number of particles, measured by light scatter on the flow cytometer, dropped significantly, showing that bacterial lysis resulted. The peptide was shown to bind to E. coli O111 lipopolysaccharide by using surface plasmon resonance. Transmission electron microscopy revealed bacterial distortion, with the outer membrane becoming detached from the inner cytoplasmic membrane. We conclude that HLP 2 causes membrane disruption of the outer membrane, resulting in lysis, and that structural considerations are important for antibacterial activity.     

Characterization of the conformations of antigenic peptides of protein lactate dehydrogenase (LDH-C4) by electrospray ionization mass spectrometry

The conformations of several rationally designed antigenic peptides that mimic, to varying degrees, an antibody-binding region of protein lactate dehydrogenase isozyme (LDH-C4) are investigated by deuterium/hydrogen exchange and electrospray ionization mass spectrometry (ESI-MS). The approach involves monitoring the reverse-exchange of deuterium, incorporated at the labile sites in the peptides, with hydrogen as a function of time by ESI-MS. Idealized forms of a segment of the native antigen are shown to be more conformationally restricted than the native peptide based on level of deuterium that remains incorporated at the labile sites over time. From the number of amide groups of the peptide backbone that retain deuterium, estimates of the helical content of each peptide have been measured that are in close agreement with those determined by Fourier transform infrared (FTIR) spectroscopy in separate experiments. A single amino acid substitution in the idealized helical construct results in a conformational change easily detected by the deuterium exchange ESI-MS method. The approach is shown to be a viable method for characterizing the conformations of protein antigens at the local level and for screening the conformations of antigenic peptides designed to elicit optimal immune responses.     

Crystal structure of the simian immunodeficiency virus (SIV) gp41 core: conserved helical interactions underlie the broad inhibitory activity of gp41 peptides

The gp41 subunit of the envelope protein complex from human and simian immunodeficiency viruses (HIV and SIV) mediates membrane fusion during viral entry. The crystal structure of the HIV-1 gp41 ectodomain core in its proposed fusion-active state is a six-helix bundle. Here we have reconstituted the core of the SIV gp41 ectodomain with two synthetic peptides called SIV N36 and SIV C34, which form a highly helical trimer of heterodimers. The 2.2 A resolution crystal structure of this SIV N36/C34 complex is very similar to the analogous structure in HIV-1 gp41. In both structures, three N36 helices form a central trimeric coiled coil. Three C34 helices pack in an antiparallel orientation into highly conserved, hydrophobic grooves along the surface of this coiled coil. The conserved nature of the N36-C34 interface suggests that the HIV-1 and SIV peptides are functionally interchangeable. Indeed, a heterotypic complex between HIV-1 N36 and SIV C34 peptides is highly helical and stable. Moreover, as with HIV-1 C34, the SIV C34 peptide is a potent inhibitor of HIV-1 infection. These results identify conserved packing interactions between the N and C helices of gp41 and have implications for the development of C peptide analogs with broad inhibitory activity.     

Purification of calcitonin-like peptides from rat brain and pituitary

Accumulating evidence supports the existence of nonthyroidal calcitonin (CT)-like peptides, more similar to fish CTs, which may act as endogenous regulators of CT receptors in brain and other tissues. In this study, we have carried out large-scale extractions from Sprague-Dawley rat brain diencephalon and pituitary, and purified a novel, biologically active, CT-like peptide from pituitary. Monitoring of the calcitonin-like activity of the peptides from rat brain and pituitary required different detection systems. While the brain CT cross-reacted with C-terminally directed salmon CT-specific antisera, the pituitary CT did not. However, the pituitary CT was biologically active, exhibiting specific interaction with CT receptors to activate adenylate cyclase. Conventional chromatographic techniques were employed to purify the CT-like peptides. Although the brain CT was not purified to homogeneity, size exclusion chromatography revealed the presence of multiple molecular weight forms of immunoreactive CT. Of these, only the lowest molecular weight form was biologically active. Purification from the pituitary resulted in the isolation of a biologically active peptide with a mass of 3267 Da. This mass differs from the mass of both salmon and thyroid-derived rat CT. Initial amino acid sequencing of the pituitary CT indicated that it was N-terminally blocked. Following aminopeptidase digestion, a unique six amino acid sequence, EKSQSP, was identified. Elucidation of the amino acid composition provided supporting evidence that the peptide was novel and was consistent with a full length peptide of approximately 30 amino acids. These data support the existence of novel, nonthyroidal, CTs which are potential regulators of CT receptor-mediated functions.     

Molecular mechanisms of calmodulin's functional versatility

Calmodulin (CaM) is a primary Ca2+-binding protein found in all eukaryotic cells. It couples the intracellular Ca2+ signal to many essential cellular events by binding and regulating the activities of more than 40 different proteins and enzymes in a Ca2+-dependent manner. CaM contains two structurally similar domains connected by a flexible central linker. Each domain of the protein binds two Ca2+ ions with positive cooperativity. The binding of Ca2+ transforms the protein into its active form through a reorientation of the existing helices of the protein. The two helices in each helix-loop-helix Ca2+-binding motif are almost antiparallel in Ca2+-free CaM. The binding of Ca2+ induces concerted helical pair movements and changes the two helices in each Ca2+ binding motif to a nearly perpendicular orientation. These concerted helix pair movements are accompanied by dramatic changes on the molecular surface of the protein. Rather than exhibiting a flat, hydrophilic molecular surface as seen in Ca2+-free CaM, the Ca2+-saturated form of the protein contains a Met-rich, cavity-containing hydrophobic surface in each domain. These hydrophobic surfaces are largely responsible for the binding of CaM to its targets. The unique flexibility and high polarizability of the Met residues located at the entrance of each hydrophobic pocket together with other hydrophobic amino acid residues create adjustable, sticky interaction surface areas that can accommodate CaM's targets, which have various sizes and shapes. Therefore, CaM is able to bind to a large array of targets without obvious sequence homology. Upon binding to its target peptides, the unwinding of the central linker allows the two domains of the protein to engulf the hydrophobic face of target peptides of differing lengths. The binding of Ca2+ reduces the backbone flexibility of CaM. Formation of complexes with its target peptides further decreases the backbone motion of CaM.     

Long-term results of total penile reconstruction with a prefabricated lateral arm free flap

Trifluoroethanol (TFE) is often used to increase the helicity of peptides to make them usable as models of helices in proteins. We have measured helix propensities for all 20 amino acids in water and two concentrations of trifluoroethanol, 15 and 40% (v/v) using, as a model system, a peptide derived from the sequence of the alpha-helix of ribonuclease T1. There are three main conclusions from our studies. (1) TFE alters electrostatic interactions in the ribonuclease T1 helical peptide such that the dependence of the helical content on pH is lost in 40% TFE. (2) Helix propensities measured in 15% TFE correlate well with propensities measured in water, however, the correlation with propensities measured in 40% TFE is significantly worse. (3) Propensities measured in alanine-based peptides and the ribonuclease T1 peptide in TFE show very poor agreement, revealing that TFE greatly increases the effect of sequence context.     

Folding propensities of peptide fragments of myoglobin

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.     

Trypsin-catalyzed peptide synthesis and various p-guanidinophenyl esters as acyl donors

Trypsin-catalyzed peptide synthesis has been studied by using p-guanidinophenyl esters of N alpha-(tert-butyloxycarbonyl)amino acid and peptide as acyl donor components. The reaction conditions were optimized for organic solvents, pH, and concentration of acceptor. The method was especially useful for the preparation of various peptides containing D-amino acids. The enzymatic hydrolysis of the resulting products was negligible.     

Specificity of Pseudomonas aeruginosa serralysin revisited, using biologically active peptides as substrates

The characterization of the specificity of alkaline protease from Pseudomonas aeruginosa has not yet been clearly defined. Some previous results suggested that its specificity was influenced more by amino acids far from the hydrolyzed peptide bond, than by amino acids in P1 or P'1 position. From other data, it was a C-(COOH)-type endoprotease where the preferential amino acid in P1 position was an arginine residue. We have studied the hydrolysis of several biologically active peptides. Many various sites of cleavage have been characterized but no arginine in P1 position was found, despite the presence of arginine in the peptide sequence. In fact P1 and P'1 position could be occupied by various amino acids. It seems unlikely that Pseudomonas alkaline protease may only be considered as a protease specific to arginine in P1 position. On the other hand, we have observed that increase of the peptide chain length led to an important increase of the hydrolysis rate, suggesting an extended number of subsites.     

Contribution of the hydrophobicity gradient of an amphipathic peptide to its mode of association with lipids

A class of peptides that associate with lipids, known as oblique-orientated peptides, was recently described [Brasseur R., Pillot, T., Lins, L., Vandekerckhove, J. & Rosseneu, M. (1997) Trends Biochem. Sci. 22, 167-171]. Due to an asymmetric distribution of hydrophobic residues along the axis of the alpha-helix, such peptides adopt an oblique orientation which can destabilise membranes or lipid cores. Variants of these oblique peptides, designed to have an homogeneous distribution of hydrophobic and hydrophilic residues along the helical axis, are classified as regular amphipathic peptides. These peptides are expected to lie parallel to the polar/apolar interface with their hydrophobic residues directed towards the apolar and their hydrophilic residues towards the polar phase. An hydrophobic, oblique-orientated peptide was identified at residues 56-68 in the sequence of the lecithin-cholesterol acyltransferase (LCAT), enzyme. This peptide is predicted to penetrate a lipid bilayer at an angle of 40 degrees through its more hydrophobic C-terminal end and thereby induce the destabilisation of a membrane or a lipid core. The LCAT-(56-68) wild-type peptide was synthesised together with the LCAT-(56-68, 0 degrees) variant, in which the hydrophobicity gradient was abolished through residue permutations. In two other variants, designed to keep their oblique orientation, the W61 residue was shifted either towards the more hydrophilic N-terminal at residue 57, or to position 68 at the hydrophobic C-terminal end of the peptide. Peptide-induced vesicle fusion was demonstrated by fluorescence measurements using pyrene-labeled vesicles and by monitoring of vesicle size by gel filtration. The interaction between peptides and lipids was monitored by measurement of the intrinsic tryptophan fluorescence emission of the peptides. Fluorescence polarisation measurements, using diphenyl hexatriene, were carried out to follow changes in the lipid fluidity. The LCAT-(56-68) wild-type peptide and the two oblique variants, induced fusion of unilamellar dimyristoylglycerophosphocholine vesicles. Tryptophan fluorescence emission measurements showed a 12-14 nm blue shift upon addition of the wild-type peptide and of the W61-->68 variant to lipids, whereas the fluorescence of the W61-->57 variant did not change significantly. This observation supports the insertion of the more hydrophobic C-terminal residues into the lipid phase, as predicted by the theoretical calculations. In contrast, the 0 degrees variant peptide had no fusogenic activity, and it associated with lipids to form small discoidal lipid/peptide complexes. The phospholipid transition temperature was decreased after addition of the wild-type, the W61-->68 and W61-->57 fusogenic peptides, whereas the opposite effect was observed with the 0 degrees variant. The behaviour of the wild-type and variant LCAT-(56-68) peptides stresses the contribution of the hydrophobicity gradient along the axis of an amphipathic peptide to the mode of association of this peptide with lipids. This parameter consequently influences the structural modifications occurring to lipids upon association with amphipathic peptides.     

The helix-hinge-helix structural motif in human apolipoprotein A-I determined by NMR spectroscopy

The conformation of a synthetic peptide of 46 residues from apoA-I was investigated by fluorescence, CD, and 2D NMR spectroscopies in lipid-mimetic environments. ApoA-I(142-187) is mainly unstructured in water but helical in SDS or dodecylphosphocholine (DPC), although the peptide only associates with DPC at approximately the critical micellar concentration. Solution structures of apoA-I(142-187) were determined by distance geometry calculations based on 450 (in DPC-d38) or 397 (in SDS-d25) NOE-derived distance restraints, respectively. Backbone RMSDs for superimposing the two helical regions 146-162 and 168-182 are 0.98 +/- 0.22 (2.38 +/- 0.20) and 1.99 +/- 0.42 (2.02 +/- 0.21) A in DPC (SDS), respectively. No interhelical NOE was found, suggesting that helix-helix interactions between the two helical domains in apoA-I(142-187) are unlikely. Similar average, curved helix-hinge-helix structures were found in both SDS and DPC micelles with the hydrophobic residues occupying the concave face, indicating that hydrophobic interactions dominate. Intermolecular NOESY experiments, performed in the presence of 50% protonated SDS, confirm that the two amphipathic helices and Y166 in the hinge all interact with the micelle. The involvement of Y166 in lipid binding is supported by fluorescence spectroscopy as well. On the basis of all the data above, we propose a model for the peptide-lipid complexes wherein the curved amphipathic helix-hinge-helix structural motif straddles the micelle. The peptide-aided signal assignment achieved for apoA-I(122-187) (66mer) and apoA-I suggests that such a structural motif is retained in the longer peptide and most likely in the intact protein.     

Helix-stabilizing nonpolar interactions between tyrosine and leucine in aqueous and TFE solutions: 2D-1H NMR and CD studies in alanine-lysine peptides

Interactions between side chains spaced (i,i + 3) and (i,i + 4) may explain the context dependence of helix propensities observed in different systems. Nonpolar residues with these spacings occur frequently in protein helices and stabilize isolated peptide helices. Here (i,i + 3) and (i,i + 4) nonpolar interactions between Tyr and Leu in different solution conditions are studied in detail in alanine-based peptides using 2D 1H NMR and CD spectroscopy. Helix contents analyzed using current models for helix-coil transitions yield interaction energies which demonstrate significant helix stabilization in aqueous 1 M NaCl solutions by Tyr-Leu or Leu-Tyr pairs when spaced (i,i + 4) and, to a smaller extent, when spaced (i,i + 3), comparable to those estimated for other residue pairs. The interactions persist in solutions containing TFE, a helix-stabilizing solvent believed to diminish hydrophobic interactions, but not in helix-destabilizing 6 M urea. 1H NMR resonances for all peptides and solution conditions except in 6 M urea were completely assigned. NMR data indicate that the N-terminal residues are more helical and that the N-acetyl group participates in helix formation. The two (i,i + 4) spaced pairs show the same pattern of NOE cross-peaks between the Tyr and Leu side chains, as do the two (i,i + 3) pairs in 1 M NaCl as well in TFE solutions, and correspond well with that expected for the specific Tyr-Leu pair with side-chain contacts in protein helices.