Helix-forming Peptides Research Articles

Uncoupling the Folding-Function Paradigm of Lytic Peptides to Deliver Impermeable Inhibitors of Intracellular Protein-Protein Interactions.

Here, we describe the use of peptide backbone N-methylation as a new strategy to transform membrane-lytic peptides (MLPs) into cytocompatible intracellular delivery vehicles. The ability of lytic peptides to engage with cell membranes has been exploited for drug delivery to carry impermeable cargo into cells, but their inherent toxicity results in narrow therapeutic windows that limit their clinical translation. For most linear MLPs, a prerequisite for membrane activity is their folding at cell surfaces. Modification of their backbone with N-methyl amides inhibits folding, which directly correlates to a reduction in lytic potential but only minimally affects cell entry. We synthesized a library of N-methylated peptides derived from MLPs and conducted structure-activity studies that demonstrated the broad utility of this approach across different secondary structures, including both β-sheet and helix-forming peptides. Our strategy is highlighted by the delivery of a notoriously difficult class of protein-protein interaction inhibitors that displayed on-target activity within cells.

Computer simulations of helix folding in homo- and heteropeptides

We present the results of microsecond length simulations of folding for three model helix-forming peptides, penta-alanine Ac-A5-NH2 (acA5a), a modified pentapeptide Ac-WA3H+-NH2 (WH5) and a 21-residue peptide Ac-WA3H+-(AAARA)3-A-NH2 (WH21), employing three protein force fields, AMBER03, CHARMM27 and OPLS-AA. The calculated helix populations are in good agreement with observations for all peptides, and the measured kinetic rate constants of WH5 are well reproduced. Our results yield new and interesting insights into the mechanism of α-helix folding. For acA5a, representing a flexible generic helical nucleus, we find significant variation of the folding pathway with employed force field. For the WH5 helix, which includes a stabilising side chain interaction, the nucleation step also varies with force field, but an intermediate with first two hydrogen bonds formed is a common feature in all models. For WH21 the three employed force fields differed in details of the helix initiation stage. However, the main stages of folding, involving first the central region, then the N-terminal and finally the C-terminal, were very similar in AMBER03, CHARMM27 and OPLS-AA. Single-helix structures were dominant intermediates, with a minor contribution from helix-turn-helix conformers. The agreement between simulations and experimental data indicate that current protein force fields provide a reliable description α-helix folding.

Effect of Hydrophobic Peptide Sequence upon Peptide-Dependent Acceleration of Lipid Flip-Flop

The asymmetric distribution of different lipid species in the inner and outer half of the bilayer is an important feature of many natural membranes. Certain transmembrane (TM) helix-forming peptides are known to increase the transverse diffusion (flip-flop) of lipids and can break down lipid asymmetry. We have investigated the dependence of peptide enhanced flip-flop upon TM helix sequence in order to try to understand the mechanism by which this occurs.The movement of a fluorescent phosphatidylcholine analog (6-NBD-PC) from the outer to inner leaflet was used to measure the influence of different model TM helices on the natural flip-flop rate of model membranes. The most hydrophobic TM peptides had very little or no effect upon flip-flop rate. Preliminary results indicate that for TM peptides of comparable length, a reduction of hydrophobicity increases the lipid flip-flop rate. The lipid flip-flop rate was most markedly increased when an Asp residue was introduced near the center of a highly hydrophobic sequence. The flip-flop was greatly accelerated when the Asp residue was protonated and the peptide was in a TM configuration but accelerated the most when the Asp residue was deprotonated and the peptide resided on the bilayer surface. Although certain pore forming peptides have been shown to accelerate flip-flop, no pore formation was detected for the peptides used in our studies. We conclude that perturbation of lipid packing is a possible mechanism by which hydrophobic helices accelerate flip-flop, while pore formation is unlikely to be a universal mechanism for accelerating flip-flop.

Probing the Kinetic Network of Folding-Unfolding Transitions in Proteins

The formation of secondary and tertiary structure elements in protein folding are intrinsically complex processes, notoriously difficult to study in a systematic manner. We construct coarse master equations for helix formation processes based on data from atomistic molecular dynamics simulations of helix-rich proteins. By carefully controlling the effects of fast, non-Markovian transitions, on one hand, and the typically limited sampling of slow relaxation processes on the other hand, we probe the underlying network of folding-unfolding transitions between the various configuration states of a protein. This systematic analysis reveals the transition states and the associated folding pathways at multiple levels, from atomistic to coarse-grained representations. We validate our approach in folding studies of short helix-forming polyalanine peptides, as well as of a larger, helix-turn-helix subdomain of a viral scaffolding protein. Our analysis of local, site-specific formation of intra- and inter-chain interactions is a first step towards understanding the elementary stages of secondary and tertiary structure formation in the folding of large proteins, and it allows a direct comparison to data from recent infrared vibrational spectroscopy studies.

Dynamics of Intra- and Inter-Helix Contact Formation

The ubiquitous nature of the helical fold and its characteristic physical properties make it one of the first candidates for studies of secondary structure formation in polypeptides, and the thermodynamics of helix formation is a common topic in many classical biophysics textbooks. However, though there is general agreement on the features of the equilibrium properties of the coil-to-helix transition, both experimental and theoretical studies have provided widely varying estimates of helix formation rates from tens of picoseconds to microseconds. We present results of recent molecular simulations of several helix-forming peptides that permit the quantitative study of both intra- and inter-helical contacts in polypeptides. This analysis of local, site-specific formation of intra- and inter-chain interactions is necessary for any quantitative modeling of the elementary steps of secondary and tertiary structure formation in protein folding, and it allows direct comparison to data from recent infrared vibrational spectroscopy studies.

Dipole-induced self-assembly of helical β-peptides

In this work, the interactions between beta-peptides are investigated for helix-forming peptides using molecular simulation. The role of electrostatic interactions in the self-assembly of these peptides is studied by calculating the dipole moment of various 14-helical beta-peptides using molecular dynamics simulations. The stability of a beta-peptide that is known to form a liquid crystalline phase is determined by calculating the potential of mean force using the expanded ensemble density of states method. This peptide is found to form a mechanically stable 14-helix in an implicit solvent model. The interaction between two of these peptides is examined by calculating the potential of mean force between the two peptides in implicit solvent. The peptides are shown to favorably associate in an end-to-end manner, driven largely by dipolar interactions. In order to understand the possible structures that form when many peptides interact in solution, a coarse-grained model is developed. Brownian dynamics simulations of the coarse-grained model at intermediate concentrations (1-50 mM) are performed, and the aggregation behavior is understood by calculating the diffusivity and the radial distribution function. An analysis of the resultant structures reveals that the coarse-grained model of the peptide leads to the formation of spherical clusters.

Improving the Oral Efficacy of Recombinant Granulocyte Colony-Stimulating Factor and Transferrin Fusion Protein by Spacer Optimization

To improve the oral efficacy of the recombinant fusion protein containing granulocyte colony-stimulating factor (G-CSF) and transferrin (Tf) by inserting a linker between the two protein domains. Oligonucleotides encoding flexible and helix-forming peptides were inserted to the recombinant plasmids. The fusion protein without linker insertion was used for comparison. The G-CSF cell-proliferation and Tf receptor-binding activities of the fusion proteins were tested in NFS-60 cells and Caco-2 cells, respectively, and in vivo myelopoietic assay with both subcutaneous and oral administration was performed in BDF1 mice. All fusion proteins produced from transfected HEK293 cells were positive in Western-blotting assay with anti-G-CSF and anti-Tf antibodies. Among them, the fusion protein with a long helical (H4-2) linker showed the highest activity in NFS-60 cell proliferation assay, with an EC50 about ten-fold lower than that of the non-linker fusion protein. The fusion protein with H4-2 linker also showed a significantly higher myelopoietic effect when administered either subcutaneously or orally in BDF1 mice. The insertion of a linker peptide, such as the helix linker H4-2, between G-CSF and Tf domains in the recombinant fusion protein can improve significantly both in vitro and in vivo myelopoietic activity over the non-linker fusion protein.

The conformation of tetraalanine in water determined by polarized Raman, FT-IR, and VCD spectroscopy.

The present article reports the conformation of cationic tetraalanine in aqueous solution. The determination of the dihedral angles of the two central amino acid residues was achieved by analyzing the amide I' band profile in the respective polarized visible Raman, Fourier transform-IR, and vibrational circular dichroism (VCD) spectra by means of a novel algorithm which utilizes the excitonic coupling between the amide I modes of nearest neighbor and second nearest peptide groups. It is an extension of a recently developed theory (Schweitzer-Stenner, R. Biophys. J., 2002, 83, 523-532). UV electronic circular dichroism (ECD) spectra of the peptides were used to validate the results of the structure analysis. The analyses yielded the dihedral angles (phi(12), psi(12)) = (-70 degrees, 155 degrees ) and (phi(23), psi(23)) = (-80 degrees, 145 degrees ). The obtained values are very close to the Ramachandran coordinates of the polyproline II helix (PPII). The data suggest that this is the conformation predominantly adopted by the peptide at room temperature. This notion was corroborated by the corresponding electronic circular dichroism spectrum. Tetraalanine exhibits a higher propensity for PPII than trialanine for which a 50:50 mixture of polyproline II and an extended beta-strand-like conformation was obtained from recent spectroscopic studies (Eker et al., J. Am. Chem. Soc. 2002, 124, 14330-14341). The temperature dependence of the CD spectra rule out that any cooperativity is involved in the strand if PPII transition. This led to the conclusion that solvent-peptide interactions give rise to the observed PPII stability. Our result can be utilized to understand why the denaturation of helix-forming peptides generally yields a PPII rather than a heterogeneous random conformation.

Exploring Flory's isolated-pair hypothesis: statistical mechanics of helix-coil transitions in polyalanine and the C-peptide from RNase A.

To evaluate Flory's isolated-pair hypothesis in the context of helical peptides, we explore equilibrium conformations of alpha-helix-forming polypeptides as a function of temperature by means of replica exchange molecular dynamics in conjunction with the CHARMM/GB implicit solvent force field and the weighted histogram analysis method. From these simulations, Zimm-Bragg parameters, s and sigma, of Ac-Alan-NMe are computed as a function of temperature. The values obtained for s(T) and sigma(T) remain unchanged along the length of the polypeptide except for very short chains and yield results consistent with measurements based on short helix-forming peptides but suggest larger s values than anticipated from polymer-based measurements. From direct estimates of the density of states for Ac-Alan-NMe (n = 3-20) and peptide constructs based on the C peptide from RNase A, the conformational entropy is calculated versus temperature. The calculated S(T) shows a clear proportionality to the chain length over a wide range of temperature. This is observed in polypeptides with both significantly branched and simple methyl (alanine) side chains. These results provide evidence for the validity of Flory's isolated pair hypothesis, at least in the context of helical peptides and helix-to-coil transitions in these peptides.

The effect of interactions involving ionizable residues flanking membrane-inserted hydrophobic helices upon helix-helix interaction.

We examined the effect of ionizable residues at positions flanking the hydrophobic core of helix-forming polyLeu peptides upon helix-helix interactions within model membrane vesicles composed of dioleoylphosphatidylcholine. The peptides studied were flanked on both the N and C termini either by two Lys (K(2)-flanked peptide), one Lys plus one Asp (DK-flanked peptide), or one Lys plus three Asp (KD(3)-flanked peptide). The fluorescence of a Trp residue positioned at the center of the hydrophobic sequence was used to evaluate peptide behavior. As judged by the concentration dependence of the maximum wavelength of Trp emission, there was significant oligomerization of the KD(3)- and DK-flanked peptides, but not the K(2)-flanked peptide, at neutral pH. At neutral pH mixtures of K(2)- and KD(3)-flanked peptides associated with each other, but mixtures of the K(2)- and DK-flanked peptides did not. Oligomerization by the DK- and KD(3)-flanked peptides decreased under low pH conditions in which the Asp residues were protonated. Additional experiments showed that at neutral pH the KD(3)-flanked peptide showed an increased tendency to oligomerize when as little as 10-15 mol % of an anionic lipid, phosphatidylglycerol, was present. The behavior of the other peptides was not strongly influenced by phosphatidylglycerol. These results can largely be explained by modulation of helix-helix interactions via electrostatic interactions involving the helix-flanking ionizable residues. Such interactions may influence membrane protein folding. The self-association of anionic KD(3)-flanked peptides suggests that additional interactions involving charged residues also can modulate helix-helix association.

Interaction of the membrane-inserted diphtheria toxin T domain with peptides and its possible implications for chaperone-like T domain behavior.

The T domain of diphtheria toxin is believed to aid the low-pH-triggered translocation of the partly unfolded A chain (C domain) through cell membranes. Recent experiments have suggested the possibility that the T domain aids translocation by acting as a membrane-inserted chaperone [Ren, J., et al. (1999) Science 284, 955-957]. One prediction of this model is that the membrane-inserted T domain should be able to interact with sequences that mimic unfolded proteins. To understand the basis of interaction of the membrane-inserted T domain with unfolded polypeptides, its interaction with water-soluble peptides having different sequences was studied. The membrane-inserted T domain was able to recognize helix-forming 23-residue Ala-rich peptides. In the presence of such peptides, hydrophobic helix 9 of the T domain underwent the previously characterized conformational change from a state exhibiting shallow membrane insertion to one exhibiting deep insertion. This conformational change was more readily induced by the more hydrophobic peptides that were tested. It did not occur at all in the presence a hydrophilic peptide in which alternating Ser and Gly replaced Ala or in the presence of unfolded hydrophilic peptides derived from the A chain of the toxin. Interestingly, a peptide with a complex sequence (RKE(3)KE(2)LMEW(2)KM(2)SETLNF) also interacted with the T domain very strongly. We conclude that the membrane-inserted T domain cannot recognize every unfolded amino acid sequence. However, it does not exhibit strong sequence specificity, instead having the ability to recognize and interact with a variety of amino acid sequences having moderate hydrophobicity. This recognition was not strictly correlated with the strength of peptide binding to the lipid, suggesting that more than just hydrophobicity is involved. Although it does not prove that the T domain functions as a chaperone, T domain recognition of hydrophobic sequences is consistent with it having polypeptide recognition properties that are chaperone-like.

Design of the linkers which effectively separate domains of a bifunctional fusion protein.

With the aim of separating the domains of a bifunctional fusion protein, the ability of several lengths of helix-forming peptides to separate two weakly interacting beta-can domains was compared with that of flexible linkers or of a three alpha-helices bundle domain. We introduced helix-forming peptide linkers A(EAAAK)nA (n = 2-5) between two green fluorescent protein variants, EBFP and EGFP, and investigated their spectral properties. The fluorescence resonance energy transfer from EBFP to EGFP decreased as the length of the linkers increased. The circular dichroism spectra analysis suggested that the linkers form an alpha-helix and the alpha-helical contents increased as the length of the linkers increased. The results clearly suggested the ability of the helical linkers to control the distance and reduce the interference between the domains. This 'linker engineering' may open a way to the rational design of linkers which maximize the multiple functions of fusion proteins or de novo multi-domain proteins.

The Effects of Chain Length and Thermal Denaturation on Helix-Forming Peptides: A Mode-Specific Analysis Using 2D FT-IR

The major goal of this project was to use FT-IR spectroscopy to monitor the effects of chain length and temperature on small, helix-forming peptides of the general form, Ac-W(EAAAR)nA-NH2, where n ...

Recognition of α-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 α-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.

Purification of Antimicrobial Peptides from an Extract of the Skin of Xenopus laevis Using Heparin-Affinity HPLC: Characterization by Ion-Spray Mass Spectrometry

A simple scheme was developed for the rapid purification of antimicrobial peptides from the skin of Xenopus laevis. An extract of the frog skin was prepared using an acidic medium designed to maximize the solubilization of low-molecular-weight peptides. This extract was subjected to an enrichment procedure using C18 Sep Pak cartridges to yield a salt-free fraction, devoid of high-molecular-weight proteins. This fraction was in turn subjected to heparin affinity high-performance liquid chromatography on a Shodex AF-Pak column. All the antibacterial activity bound to the column and could be eluted using a linear gradient of increasing sodium chloride concentration. Antibacterial activity emerged from the column in fractions corresponding to a sodium chloride concentration of 0.45 M. Reversed-phase high-performance liquid chromatography resolved this material into a series of compounds which could be readily characterized using a combination of amino acid analysis and ion-spray mass spectrometry. Each peptide was found to be antimicrobial and each was positively identified as belonging to a family of amphipathic helix-forming peptides characterized by other investigators. Listed in their order of elution from the reversed-phase column the peptides were magainin 2, magainin 1, peptide-glycine-leucine amide, xenopsin precursor fragment, levitide precursor fragment, and a mixture of fragments derived from the caerulein precursor. These peptides owe their antimicrobial properties to a predeliction to forming amphipathic α-helical structures when associated with lipid membranes. The distribution of lysine residues within their sequences, while facilitating amphipathic helix formation, most likely also facilitates their binding to heparin. The basic peptides, xenopsin, levitide and metabolic fragments of magainin 2, were not bound by the heparin column. This suggested that the affinity of the antibacterial peptides for the affinity column is not solely a cation exchange phenomenon. A novel cystine-rich peptide 66 residues in length was also discovered in the nonretained fraction. Sequence analysis revealed that this peptide was structurally related to the neurotoxin/cytotoxin family previously found only in the venom of certain species of poisonous snakes. This study demonstrates how heparin-affinity chromatography can be utilized to purify basic amphipathic paptides and should prove useful in isolating similar antibacterial peptides from other sources such as the intestinal and pulmonary epithelial tissues of higher vertebrates.

Rotational diffusion and intermolecular collisions of a spin labeled alpha-helical peptide determined by electron spin echo spectroscopy.

Short peptides that are composed mainly of alanine have recently been shown to form alpha-helices in aqueous solution at low temperature (Marqusee, S., and R. L. Baldwin. 1987. Proc. Natl. Acad. Sci. 84:8898-8902; Marqusee, S., V. H. Robbins, and R. L. Baldwin. 1989. Proc. Natl. Acad. Sci. USA. 86:5286-5290). These peptides are excellent models for probing structure and dynamics in isolated helical domains. In previous work we have designed and synthesized spin labeled analogs of these helix-forming peptides and we have shown that these analogs retain the folding characteristics of the parent peptide (Todd, A. P., and G. L. Millhauser. 1991. Biochemistry. 30:5515-5523). Using conventional continuous wave electron spin resonance (CW ESR) we have further shown that local motion is more pronounced near the helix amino terminus than in the central region as the peptide is thermally unfolded (Miick, S. M., A. P. Todd, and G. L. Millhauser. 1991. Biochemistry. 30:9498-9503). In this present work we use electron spin echo (ESE) spectroscopy to further refine our understanding of the solution dynamics of the 3K-8 peptide, which is a 16-mer with a nitroxide spin label attached at position 8. We find that the spin echo decays are well described by a single exponential function and that the determined correlation times are close to those previously derived from CW experiments. Variable concentration ESE experiments have directly revealed Heisenberg spin exchange (HSE) interactions and we find that the interpeptide collision rate is near to that expected for a free species in solution. This provides strong evidence that the helical conformation of these peptides is not stabilized by intermolecular interactions.

Purification of recombinant pp60v-src protein tyrosine kinase and phosphorylation of peptides with different secondary structure preference

The expression of the transforming gene product of Rous sarcoma virus (pp60v-src) in Saccharomyces cerevisiae has recently been reported (Kornbluth et al., 1987; Brugge et al., 1987). To carry out biochemical and structural studies of this enzyme, a facile purification was developed. The purification was accomplished in four chromatographic steps: Q-Sepharose, Affi-Gel Blue, phosphoagarose, and hydroxylapatite chromatography. The tyrosine kinase was isolated in milligram quantities as two highly active proteolytic fragments (52 and 54 kDa). Three model tyrosine kinase substrates with propensities to adopt helical or omega-loop conformations were synthesized and characterized. The peptides were based on the sites of phosphorylation of pp60v-src, lipocortin I, and lipocortin II. Circular dichroism spectroscopy was used to study the conformation of the helix-forming peptides in 50 mM Tris and in 50% trifluoroethanol/Tris. Peptide 1, which was designed to form an amphiphilic alpha-helix, displayed 24.2% helicity in buffer and 40.2% helicity in 50% TFE/buffer. Similar experiments for peptide 3, the other helix former, showed a lower helicity (8.1% helical and 26.0% helical in buffer and in 50% TFE/buffer, respectively). All three peptides were shown to be substrates for the recombinant tyrosine kinase. Kinetic measurements using high-voltage paper electrophoresis indicated that the helix-forming peptides exhibited low KM values (approximately 450 microM) for the purified src gene product, consistent with the notion that elements of secondary structure may be important in substrate recognition by tyrosine kinases.

Properties of lipid complexes with amphipathic helix-forming peptides: Role of distribution of peptide charges.

The peptides [Glu1,8,Leu11,17] 18A and [Glu4,9,Leu11,17] reverse-18A are 18-residue peptides designed to form amphipathic helices with opposite charge distribution; [Glu1,8,Leu11,17] 18A having positively charged residues at the hydrophobic/hydrophilic interface. Both [Glu1,8,Leu11,17] 18A and [Glu4,9,Leu11,17] reverse-18A strongly disrupt the bilayer structure as indicated by the relatively narrow lipid 1H and 31P NMR peaks. In addition, the 1H chemical shift of the quaternary ammonium methyl groups indicates that [Glu1,8,Leu11,17] 18A forms smaller lipoprotein particles with dimyristoylphosphatidylcholine (DMPC) than does [Glu4,9,Leu11,17] reverse-18A. However, motional properties of the lipid head group indicate that no specific salt bridges are formed between the phospholipid head group and the side chains of polar amino acids of either of the two peptides. In addition, the acyl chain conformation for the DMPC complexes with [Glu1,8,Leu11,17] 18A and with [Glu4,9,Leu11,17] reverse-18A are indistinguishable by the criterion of IR spectroscopy. The 2H linewidth of the solvent 2H2O remains narrower in frozen solutions of the DMPC-[Glu1,8,Leu11,17] 18A complexes suggesting the presence of more unfrozen bound water in this case. The two peptides exhibit many similarities in their interaction with lipids. However, [Glu1,8,Leu11,17] 18A can more readily lyse vesicles and activate lecithin:cholesterol acyltransferase. These differences do not appear to result from differences in specific charge interactions between the lipid and peptide but may be manifested through differences in hydration properties.