α-Helix to β-sheet transition within the LeuiLysj (i = 2j) series of lytic amphipathic peptides by decreasing their size

Summary De novo designed extremely simplified amphipathic basic Leu_iLys_j (i=2j) peptides of 8, 9 and 15 residues were synthesized to clarify the mechanism of action of natural cytotoxic and hemolytic small proteins or peptides. They proved to have strong hemolytic activity towards human erythrocytes which increases with peptide length. These peptides are highly surface active and form stable peptidic films at the air/water interface. The sensitive and efficient FTIR modulated polarization technique (PMIRRAS) allows one to obtain in situ structural and orientational information about the peptides at the interface. A transition of secondary structure is observed: the shorter peptides (8 and 9 residues) adopt β-sheet structures while the longer one (15 residues) is folded into an α-helix. In both cases, the peptides lie with the axis parallel to the interface. Their insertion into a dimyristoylphosphatidylcholine monolayer can be followed from the increase in the surface and/or pressure of the films. In the mixed films, the peptides adopt the same structure and orientation as observed at the air/water interface. Therefore, among the same series of peptides, a transition from β-sheet to α-helix occurs when the length increases (roughly>10 aa), but despite this drastic change both types of structures result in strongly hemolytic peptides.     

Variants of 3(10)-helices in proteins

An analysis of the shortest 3(10)-helices, containing three helical residues and two flanking capping residues that participate in two consecutive i + 3 --> i hydrogen bonds, shows that not all helices belong to the classic 3(10)-helix, where the three central residues adopt the right-handed helical conformation (alpha(R)). Three variants identified are: 3L10-helix with all residues in the left-handed helical region (alpha(L)), 3EL10-helix where the first residue is in the extended region followed by two residues in the alpha(L) conformation, and its mirror-image, the 3E'R10-helix. In the context of these helices, as well as the equivalent variants of alpha-helices, the length dependence of the handedness of secondary structures in protein structure is discussed. There are considerable differences in the amino acid preferences at different positions in the various types of 3(10)-helices. Each type of 3(10)-helix can be thought to be made up of an extension of a particular type of beta-turn (made up of residues i to i + 3) such that the (i + 3)th residue assumes the same conformation as the preceding residue. Distinct residue preferences at i and i + 3 positions seem to decide whether a particular stretch of four residues will be a beta-turn or a 3(10)-helix in the folded structure.     

Dermaseptin S9, an alpha-helical antimicrobial peptide with a hydrophobic core and cationic termini

The dermaseptins S are closely related peptides with broad-spectrum antibacterial activity that are produced by the skin of the South American hylid frog, Phyllomedusa sauvagei. These peptides are polycationic (Lys-rich), alpha-helical, and amphipathic, with their polar/charged and apolar amino acids on opposing faces along the long axis of the helix cylinder. The amphipathic alpha-helical structure is believed to enable the peptides to interact with membrane bilayers, leading to permeation and disruption of the target cell. We have identified new members of the dermaseptin S family that do not resemble any of the naturally occurring antimicrobial peptides characterized to date. One of these peptides, designated dermaseptin S9, GLRSKIWLWVLLMIWQESNKFKKM, has a tripartite structure that includes a hydrophobic core sequence encompassing residues 6-15 (mean hydrophobicity, +4.40, determined by the Liu-Deber scale) flanked at both termini by cationic and polar residues. This structure is reminiscent of that of synthetic peptides originally designed as transmembrane mimetic models and that spontaneously become inserted into membranes [Liu, L., and Deber, C. M. (1998) Biopolymers 47, 41-62]. Dermaseptin S9 is a potent antibacterial, acting on gram-positive and gram-negative bacteria. The structure of dermaseptin S9 in aqueous solution and in TFE/water mixtures was analyzed by circular dichroism and two-dimensional NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin S9 is aggregated in water, but a monomeric nonamphipathic alpha-helical conformation, mostly in residues 6-21, is stabilized by the addition of TFE. These results, combined with membrane permeabilization assays and surface plasmon resonance analysis of the peptide binding to zwitterionic and anionic phospholipid bilayers, demonstrate that spatial segregation of hydrophobic and hydrophilic/charged residues on opposing faces along the long axis of a helix is not essential for the antimicrobial activity of cationic alpha-helical peptides.     

Identification of an apolipoprotein A-I structural element that mediates cellular cholesterol efflux and stabilizes ATP binding cassette transporter A1

Synthetic peptides were used in this study to identify a structural element of apolipoprotein (apo) A-I that stimulates cellular cholesterol efflux and stabilizes the ATP binding cassette transporter A1 (ABCA1). Peptides (22-mers) based on helices 1 (amino acids 44-65) and 10 (amino acids 220-241) of apoA-I had high lipid binding affinity but failed to mediate ABCA1-dependent cholesterol efflux, and they lacked the ability to stabilize ABCA1. The addition of helix 9 (amino acids 209-219) to either helix 1 (creates a 1/9 chimera) or 10 (9/10 peptide) endowed cholesterol efflux capability and ABCA1 stabilization activity similar to full-length apoA-I. Adding helix 9 to helix 1 or 10 had only a small effect on lipid binding affinity compared with the 22-mer peptides, indicating that helix length and/or determinants on the polar surface of the amphipathic alpha-helices is important for cholesterol efflux. Cholesterol efflux was specific for the structure created by the 1/9 and 9/10 helical combinations, as 33-mers composed of helices 1 and 3 (1/3), 2/9, and 4/9 failed to mediate cholesterol efflux in an ABCA1-dependent manner. Transposing helices 9 and 10 (10/9 peptide) did not change the class Y structure, hydrophobicity, or amphiphilicity of the helical combination, but the topography of negatively charged amino acids on the polar surface was altered, and the 10/9 peptide neither mediated ABCA1-dependent cholesterol efflux nor stabilized ABCA1 protein. These results suggest that a specific structural element possessing a linear array of acidic residues spanning two apoA-I amphipathic alpha-helices is required to mediate cholesterol efflux and stabilize ABCA1.     

Improved solid-phase synthesis of alpha,alpha-dialkylated amino acid-rich peptides with antimicrobial activity.

A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid-phase synthetic techniques. Each nonapeptide was rich in alpha,alpha-dialkylated amino acids [one 4-aminopiperidine-4-carboxylic acid (Api) and six alpha-aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9-fluorenylmethyloxycarbonyl (Fmoc)-Aib-Aib-OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3(10)-helical, amphipathic design of these peptides was born out most prominently in the N-terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations < or =100 microM) and the acetylated peptides (concentrations < or = 200 microM) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.     

Long, chiral polypeptide 3(10)-helices at atomic resolution

The crystal-state preferred conformation of the terminally blocked hepta- and octapeptides with the general formula -(Aib)n L-Leu-(Aib)2- (n = 4 and 5, respectively), determined by X-ray diffraction, was found to be a right-handed 3(10)-helix stabilized by five and six consecutive intramolecular NH...O = C H-bonds of the C(10)-III type, respectively. The octapeptide structure represents the first observation at atomic resolution of a regular, chiral 3(10)-helix larger than two complete turns. In both cases the right handed screw sense of the helix is dictated by the presence of the single, internal L-residue. This study confirms the propensity of short peptides rich in Aib, the prototype of the amino acid residues dialkylated at the alpha carbon, to adopt a 3(10)-helical structure and is expected to help our understanding of the conformational preferences of the membrane-active, channel-forming, ion-transporting peptaibol antibiotics.     

Addition of side-chain interactions to 3(10)-helix/coil and alpha-helix/3(10)-helix/coil theory.

An increasing number of experimental and theoretical studies have demonstrated the importance of the 3(10)-helix/ alpha-helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side-chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the alpha-helix, will favor the alpha-helix over 3(10), while an i, i + 4 repulsion will favor the 3(10)-helix over alpha. To quantify the 3(10)/alpha/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 3(10)-helix/coil and 3(10)-helix/alpha-helix/ coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side-chain interaction energies to the models. The theory is based on classifying residues into alpha-helical, 3(10)-helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side-chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i, i + 4 side-chain interactions favor alpha-helix formation, while the 3(10)-helix population is maximized when weaker i, i + 4 side-chain interactions are present.     

Relationship of sidechain hydrophobicity and α-helical propensity on the stability of the single-stranded amphipathic α-helix

The aim of the present investigation is to determine the effect of α-helical propensity and sidechain hydrophobicity on the stability of amphipathic α-helices. Accordingly, a series of 18-residue amphipathic α-helical peptides has been synthesized as a model system where all 20 amino acid residues were substituted on the hydrophobic face of the amphipathic α-helix. In these experiments, all three parameters (sidechain hydrophobicity, α-helical propensity and helix stability) were measured on the same set of peptide analogues. For these peptide analogues that differ by only one amino acid residue, there was a 0.96 kcal/mole difference in α-helical propensity between the most (Ala) and the least (Gly) α-helical analogue, a 12.1-minute difference between the most (Phe) and the least (Asp) retentive analogue on the reversed-phase column, and a 32.3°C difference in melting temperatures between the most (Leu) and the least (Asp) stable analogue. The results show that the hydrophobicity and α-helical propensity of an amino acid sidechain are not correlated with each other, but each contributes to the stability of the amphipathic α-helix. More importantly, the combined effects of α-helical propensity and sidechain hydrophobicity at a ratio of about 2:1 had optimal correlation with α-helix stability. These results suggest that both α-helical propensity and sidechain hydrophobicity should be taken into consideration in the design of α-helical proteins with the desired stability.     

Structural design and characterization of a channel-forming peptide

A 16-residue polypeptide model with the sequence acetyl-YALSLAATLLKEAASL-OH was derived by rational de novo peptide design. The designed sequence consists of amino acid residues with high propensity to adopt an alpha helical conformation, and sequential order was arranged to produce an amphipathic surface. The designed sequence was chemically synthesized using a solid-phase method and the polypeptide was purified by reverse-phase liquid chromatography. Molecular mass analysis by electro-spray ionization mass spectroscopy confirmed the correct designed sequence. Structural characterization by circular dichroism spectroscopy demonstrated that the peptide adopts the expected alpha helical conformation in 50% acetonitrile solution. Liposome binding assay using Small Unilamellar Vesicle (SUV) showed a marked release of entrapped glucose by interaction between the lipid membrane and the tested peptide. The channel-forming activity of the peptide was revealed by a planar lipid bilayer experiment. An analysis of the conducting current at various applied potentials suggested that the peptide forms a cationic ion channel with an intrinsic conductance of 188 pS. These results demonstrate that a simple rational de novo design can be successfully employed to create short peptides with desired structures and functions.     

Effect of terminal achiral and chiral residues on the conformational behaviour of poly Δ(z)Phe and analysis of various interactions

Conformational properties of the peptides containing (Δ(Z)Phe)6 with achiral (ΔAla, Gly) and chiral (Ala, Leu) residues at both the N- and C-terminal positions have been studied with a view to design a peptide with desired helical screw sense. In all the peptides, the lowest energy conformational state corresponds to Φ = 0° and Ψ = + 90° or - 90° or both +/- 90°. These structures are characterized by rise per residue of 1.94 ?; rotation per residue of 114° and 3.12 residues per turn and are stabilized by: (i) carbonyl-carbonyl interactions with the carbonyl oxygen of ith residue and carbonyl carbon atom of the carbonyl group of ith+1 residue; and (ii) N-H....π interactions between the amino group of Δ(Z)Phe and its own aromatic moiety. The Ala/Leu residues at the N-terminus further stabilized the structure, through C-H....π interactions with the farthest edge of the aromatic ring of ith+3 Δ(Z)Phe residue. For peptides Ac-L-Ala/L-Leu-(Δ(Z)Phe)6-NHMe, the low energy left handed helical structure (approximately 2.5 Kcalmol?1 higher in energy) state corresponds to Φ = -30°, Ψ = 120° for L-residue and Φ = Ψ = 30° for Δ(Z)Phe residues and is in good agreement with the X-ray crystallography results for the peptide Boc-L-Ala-(Δ(Z)Phe)4-NHMe crystals grown from acetonitrile/ethanol mixture. Computational results suggest that the peptides Ac-DAla/D-Leu-(Δ(Z)Phe)6-NHMe adopt a right handed helical structure in polar solvents with Φ = 30°, Ψ = -120° for D-residues and Φ = Ψ = -30° for Δ(Z)Phe residues. Both in the left handed and right handed structures, the carbonyl oxygen of acetyl group is involved in 10-membered hydrogen bonded ring formation with NH of 3rd Δ(z)Phe residue whereas Δ(Z)Phe residues backbone adopts a 3?? helix structure. Computational results also suggest that the conformational state with Φ = 0° and Ψ = 90° can be realized by keeping D-Ala or D-Leu at the C-terminal. There is hardly any effect of achiral residues Gly/ΔAla on the conformational behaviour of poly-Δ(Z)Phe.     

Design and synthesis of novel nonpolar host peptides for the determination of the 310- and α-helix compatibilities of α-amino acid buildig blocks: An assessment of α,α-disubstituted glycines

The present work describes three novel nonpolar host peptide sequences that provide a ready assessment of the 3₁₀- and α-helix compatibilities of natural and unnatural amino acids at different positions of small- to medium-size peptides. The unpolar peptides containing Ala, Aib, and a C-terminal p-iodoanilide group were designed in such a way that the peptides could be rapidly assembled in a modular fashion, were highly soluble in solvent mixtures of triflouroethanol and H₂O for CD- and two-dimensional (2D) nmr spectroscopic analyses, and showed excellent crystallinity suited for x-ray structure analysis. To validate our approach we synthesized 9-mer peptides 79a–96 (Table IV), 12-mer peptides 99–110c (Table V), and 10-mer peptides 120a–125d and 129–133 (Table VI and Scheme 8) incorporating a series of optically pure cyclic and open-chain (R)- and (S)-α,α-disubstituted glycines 1–10 (Figure 2). These amino acids are known to significantly modulate the conformations of small peptides. Based on x-ray structures of 9-mers 79a, 80, and 87 (Figures 4–7), 10-mers 124c, 131, and 132 (Figures 9–12), and 12-mer peptide 102b (Figure 13), CD spectra of all peptides recorded in acidic, neutral, and basic media and detailed 2D-nmr analyses of 9-mer peptide 86 and 12-mer 102b, several interesting conformational observations were made. Especially interesting results were obtained using the convex constraint CD analysis proposed by Fasman on 9-mer peptides 79a–d, 80, 81, 86, and 87, which allowed us to determine the relative content of 3₁₀- and α-helical conformations. These results were fully supported by the corresponding x-ray and 2D-nmr analyses. As a striking example we found that the (S)- and (R)-β-tetralin derived amino acids (R)- and (S)-1 show excellent α-helix stabilisation, more pronounced than Aib and Ala. These novel reference peptide sequences should help establish a scale for natural and unnatural amino acids concerning their intrinsic 3₁₀- and α-helix compatibilities at different positions of medium-sized peptides and thus improve our understanding in the folding processes of peptides. © 1997 John Wiley & Sons, Inc. Biopoly 42: 575–626, 1997     

Correlation between symmetry breaker position and the preferences of conformationally constrained homopeptides: a molecular dynamics investigation

The conformational tendencies of C(alpha,alpha)-diethylglycine (Deg)-based peptides have been studied in solution using all atom molecular dynamics simulations. Specifically, the conformational effects of breaking the symmetry of the host Tfa-(Deg)(5)-OtBu (Tfa, trifluoroacetyl; OtBu, tert-butoxy) pentapeptide with punctual replacements at different sequence positions of one Deg residue by its corresponding guest chiral analogue, L-alpha-aminobutyric acid (L-Abu), have been examined by simulating the following peptides: Tfa-(Deg)(5)-OtBu, Tfa-(Deg)(2)-L-Abu-(Deg)(2)-OtBu, Tfa-(Deg)(3)-L-Abu-Deg-OtBu, and Tfa-(Deg)(4)-L-Abu-OtBu. Simulations show that only the Deg homopeptide is able to stabilize a 2.0(5) helix, even though a kinked arrangement with all the Deg residues adopting a fully-extended conformation was found to be stable when the L-Abu residue is introduced in the middle of the sequence. On the other hand, when the L-Abu residue is closer to the C-end of the sequence, the peptide chain prefers a partially folded 3(10)-helix. Additional simulations on Tfa-(Deg)(3)-L-Abu-(Deg)(3)-OtBu highlighted that, when the size of the Deg segments increases, their tendency to adopt a 2.0(5) helix predominates over the preferred folded conformation of L-Abu. The overall picture extracted after more than 300 ns of molecular dynamics simulation is that breaking the alpha-carbon symmetry of achiral C(alpha)-tetrasubstituted amino acids is a promising strategy to build up polypeptides with modulated conformational tendencies.     

Three-dimensional structure of the two-peptide bacteriocin plantaricin JK

The three-dimensional structures of the two peptides, PlnJ and PlnK, that constitutes the two-peptide bacteriocin plantaricin JK have been solved in water/TFE and water/DPC-micellar solutions using nuclear magnetic resonance (NMR) spectroscopy. PlnJ, a 25 residue peptide, has an N-terminal amphiphilic α-helix between Trp-3 and Tyr-15. The 32 residues long PlnK forms a central amphiphilic α-helix between Gly-9 and Leu-24. Measurements of the effect on anti-microbial activity of single glycine replacements in PlnJ and PlnK show that Gly-13 and Gly-17 in both peptides are very sensitive, giving more than a 100-fold reduction in activity when large residues replace glycine. In variants where other glycine residues, Gly-20 in PlnJ and Gly-7, Gly-9, Gly-24 and Gly-25 in PlnK, were replaced, the activity was reduced less than 10-fold. It is proposed that the detrimental effect on activity when exchanging Gly-13 and Gly-17 in PlnJ and PlnK is a result of reduced ability of the two peptides to interact through the GxxxG-motifs constituting Gly-13 and Gly-17.     

Influence of C-terminal α-helix hydrophobicity and aromatic amino acid content on apolipoprotein A-I functionality

The apoA-I molecule adopts a two-domain tertiary structure and the properties of these domains modulate the ability to form HDL particles. Thus, human apoA-I differs from mouse apoA-I in that it can form smaller HDL particles; the C-terminal α-helix is important in this process and human apoA-I is unusual in containing aromatic amino acids in the non-polar face of this amphipathic α-helix. To understand the influence of these aromatic amino acids and the associated high hydrophobicity, apoA-I variants were engineered in which aliphatic amino acids were substituted with or without causing a decrease in overall hydrophobicity. The variants human apoA-I (F225L/F229A/Y236A) and apoA-I (F225L/F229L/A232L/Y236L) were compared to wild-type (WT) apoA-I for their abilities to (1) solubilize phospholipid vesicles and form HDL particles of different sizes, and (2) mediate cellular cholesterol efflux and create nascent HDL particles via ABCA1. The loss of aromatic residues and concomitant decrease in hydrophobicity in apoA-I (F225L/F229A/Y236A) has no effect on protein stability, but reduces by a factor of about three the catalytic efficiencies (V_max/K_m) of vesicle solubilization and cholesterol efflux; also, relatively large HDL particles are formed. With apoA-I (F225L/F229L/A232L/Y236L) where the hydrophobicity is restored by the presence of only leucine residues in the helix non-polar face, the catalytic efficiencies of vesicle solubilization and cholesterol efflux are similar to those of WT apoA-I; this variant forms smaller HDL particles. Overall, the results show that the hydrophobicity of the non-polar face of the C-terminal amphipathic α-helix plays a critical role in determining apoA-I functionality but aromatic amino acids are not required. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Mapping of a putative surface-binding site of human coagulation factor XII

We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces. A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated. Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies. Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31. Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues. These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII. Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides. Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13. We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII. It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation.     

Investigation of penetratin peptides. Part 1. The environment dependent conformational properties of penetratin and two of its derivatives.

The homeodomain, the DNA-binding domain of Antennapedia homeoprotein, is composed of three alpha-helices and one beta-turn between helices II and III. Its third helix from the N-terminal (helix III) can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. To the best of our knowledge, this helix III, called penetratin, which consists of 16 amino acids, is internalized by cells in a specific, non-receptor-mediated manner. For a better understanding of the mechanism of the transfer, the structure of penetratin was examined in both extracellular matrix-mimetic and membrane-mimetic environments: 1H-NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. The molecular conformations of two analogue peptides [(6,14-Phe)-penetratin and a 12 amino acid penetratin derivative (peptide 3)] were also studied. An atomic level comprehensive analysis of penetratin and its two analogues was performed. In a membrane-mimetic solvent system (TFEd2/water = 9: 1), on the basis of 553 distance restraints, the 4-12 region of penetratin exhibits a bent, irregular helical structure on NMR examination. Interactions between hydrophobic amino acid residues in conjunction with H-bonds stabilize the secondary structure of the molecule. Thus, both derivatives adopt a helix-like conformation. However, while (6,14-Phe)-penetratin displays both alpha-helical and 310-helical features, the structure of peptide 3 is predominantly a 310-helix. Of the three peptides, surprisingly (6,14-Phe)-penetratin has the largest helical content. An increase in the polarity of the molecular environment gradually disintegrates these helix-like secondary structures. In a highly aqueous molecular system (TFEd2/water = 1 : 9), the fast exchange of multiple conformers leads to too few distance restraints being extracted, therefore the NMR structures can no longer be determined. The NMR data show that only short-range order can be traced in these peptides. Under these conditions, the molecules adopt nascent helix-like structures. On the other hand, CD spectra could be recorded at any TFE/water ratio and the conformational interconversion could therefore be monitored as a function of the polarity of the molecular environment. The CD data were analysed comprehensively by the quantitative deconvolution method (CCA+). All three penetratin peptides display helical conformational features in a low dielectric medium, with significant differences as a function of their amino acid composition. However, these conformational features are gradually lost during the shift from an apolar to a polar molecular environment.     

A helical arrangement of beta-substituents of dehydropeptides: synthesis and conformational study of sequential nona- and dodecapeptides possessing (Z)-beta-(1-naphthyl)dehydroalanine residues

Sequential nona- and dodecapeptides possessing three and four (Z)-beta -(1-naphthyl)dehydroalanine (Delta(Z)Nap) residues, Boc-(L-Ala-Delta(Z)Nap-L-Leu)(n)-OCH(3) (n = 3 and 4; Boc = t-butoxycarbonyl), were synthesized to design a rigid 3(10)-helical backbone for a regular arrangement of functional groups using dehydropeptides. Their solution conformations were investigated by NMR and CD analyses, and theoretical energy calculations. Both peptides were found to adopt a 3(10)-helical conformation in CDCl(3) from their nuclear Overhauser effect spectroscopy (NOESY) spectra, which showed intense cross peaks for N(i)H-N(i+1)H proton pairs, but no cross peaks for C(alpha)(i)H-N(i+4)H pairs. The predominance of a 3(10)-helix was also supported by solvent accessibility of NH resonances. CD spectra of both peptides in tetrahydrofuran showed strong exciton couplets at around 228 nm assignable to naphthyl side chains, which are regularly arranged along a right-handed helical backbone. Chain-length effects on conformational preference in sequential peptide -(Ala-Delta(Z)Nap-Leu)(n)- were discussed based on spectroscopic analysis, energy minimization, and molecular dynamics simulations. Consequently, the repeating number n > or = 3 forms predominantly a right-handed 3(10)-helical conformation. The energy calculation also revealed that the midpoint naphthyl groups of peptide n = 4 are highly restricted to one stable orientation. In conclusion, beta-substituted alpha,beta-dehydroalanine is expected to be a unique tool for designing a rigid molecular frame of 3(10)-helix along which beta-functional groups are regularly arranged in a specific manner.     

De novo design and structure-activity relationships of peptide emulsifiers and foaming agents

A series of eight amphipathic peptides (8, 11, 15, 2 x 18, 22, 26, 29 amino acids in length) were designed to investigate the effects of amino acid composition, peptide length and secondary structure on surface activity assessed as emulsification and foaming activity. The potential for alpha-helix formation at the hydrophobic/hydrophilic interface was maximized through the use of helix-forming amino acids, a relatively large hydrophobic surface of 200 degrees of arc and ion pairs between basic and acidic amino acids on the hydrophilic surface. Emulsification activity increased rapidly between 11 and 22 residues as alpha-helicity in aqueous solution increased. Despite their small size, the peptides produced exceptionally stable emulsions, compared with proteins. Foaming activity was enhanced by the presence of aromatic amino acids and the activity of the best peptide examined was superior to that of bovine serum albumin and beta-lactoglobulin.     

Alpha helical structures in the leader sequence of human GLUD2 glutamate dehydrogenase responsible for mitochondrial import

Human glutamate dehydrogenase (hGDH) exists in two highly homologous isoforms with a distinct regulatory and tissue expression profile: a housekeeping hGDH1 isoprotein encoded by the GLUD1 gene and an hGDH2 isoenzyme encoded by the GLUD2 gene. There is evidence that both isoenzymes are synthesized as pro-enzymes containing a 53 amino acid long N-terminal leader peptide that is cleaved upon translocation into the mitochondria. However, this GDH signal peptide is substantially larger than that of most nuclear DNA-encoded mitochondrial proteins, the leader sequence of which typically contains 17-35 amino acids and they often form a single amphipathic α-helix. To decode the structural elements that are essential for the mitochondrial targeting of human GDHs, we performed secondary structure analyses of their leader sequence. These analyses predicted, with 82% accuracy, that both leader peptides are positively charged and that they form two to three α-helices, separated by intermediate loops. The first α-helix of hGDH2 is strongly amphipathic, displaying both a positively charged surface and a hydrophobic plane. We then constructed GLUD2-EGFP deletion mutants and used them to transfect three mammalian cell lines (HEK293, COS 7 and SHSY-5Y). Confocal laser scanning microscopy, following co-transfection with pDsRed2-Mito mitochondrial targeting vector, revealed that deletion of the entire leader sequence prevented the enzyme from entering the mitochondria, resulting in its retention in the cytoplasm. Deletion of the first strongly amphipathic α-helix only was also sufficient to prevent the mitochondrial localization of the truncated protein. Moreover, truncated leader sequences, retaining the second and/or the third putative α-helix, failed to restore the mitochondrial import of hGDH2. As such, the first N-terminal alpha helical structure is crucial for the mitochondrial import of hGDH2 and these findings may have implications in understanding the evolutionary mechanisms that led to the large mitochondrial targeting signals of human GDHs.