Antimicrobial Properties of Brevinin‐2‐Related Peptide and its Analogs: Efficacy Against Multidrug‐Resistant Acinetobacter baumannii

Brevinin‐2 related peptide (B2RP; GIWDTIKSMG¹⁰KVFAGKILQN²⁰L.NH₂), first isolated from skin secretions of the mink frog Lithobates septentrionalis, shows broad‐spectrum antimicrobial activity but its therapeutic potential is limited by moderate hemolytic activity. The peptide adopts an α‐helical conformation in a membrane‐mimetic solvent but amphipathicity is low. Increasing amphipathicity together with hydrophobicity by the substitutions Lys¹⁶→Leu and Lys¹⁶→Ala increased hemolytic activity approximately fivefold without increasing antimicrobial potency. The substitution Leu¹⁸→Lys increased both cationicity and amphipathicity but produced decreases in both antimicrobial potency and hemolytic activity. In contrast, increasing cationicity of B2RP without changing amphipathicity by the substitution Asp⁴→Lys resulted in a fourfold increase in potency against Escherichia coli [minimal inhibitory concentration (MIC) = 6 μm ) and twofold increases in potency against Staphylococcus aureus (MIC = 12.5 μm ) and Candida albicans (MIC = 6 μm ) without changing significantly hemolytic activity against human erythrocytes (LC₅₀ = 95 μm ). The emergence of antibiotic‐resistant strains of the Gram‐negative bacterium Acinetobacter baumannii constitutes a serious risk to public health. B2RP (MIC = 3–6 μm ) and [Lys⁴]B2RP (MIC = 1.5–3 μm ) potently inhibited the growth of nosocomial isolates of multidrug‐resistant Acinetobacter baumannii. Although the analogs [Lys⁴, Lys¹⁸]B2RP and [Lys⁴, Ala¹⁶, Lys¹⁸]B2RP showed reduced potency against Staphylococcus aureus, they retained activity against Acinetobacter baumannii (MIC = 3–6 μm ) and had very low hemolytic activity (LC₅₀ > 200 μm ).     

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val-Gly-Val-Lys, corresponding to sequence 296–316 of thermolysin and thus encompassing the COOH-termi-nal helical segment 301–312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296–316 was then cleaved with trypsin at Lys³⁰⁷ and Staphylococcus aureus V8 protease at Glu³⁰², producing the additional fragments 296–307, 308–316, 296–302, and 303–316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296–316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301–312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296–316 attains in 90% aqueous trifluoroethanol the same percentage (～58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296–316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule     

Solution structure of a cathelicidin‐derived antimicrobial peptide,CRAMP as determined by NMR spectroscopy

Abstract: CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin‐derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram‐positive and gram‐negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H₂O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly α‐helical conformation in TFE/H₂O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H₂O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic α‐helices from Leu⁴ to Lys¹⁰ and from Gly¹⁶ to Leu³³. These two helices are connected by a flexible region from Gly¹¹ to Gly¹⁶. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18‐residue fragment with the sequence from Gly¹⁶ to Leu³³ was found to retain antibacterial activity. Therefore, the amphipathic α‐helical region from Gly¹⁶ to Leu³³ of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.     

Determination of stereochemistry stability coefficients of amino acid side‐chains in an amphipathic α‐helix

Abstract: We describe here a systematic study to determine the effect on secondary structure of d ‐amino acid substitutions in the nonpolar face of an amphipathic α‐helical peptide. The helix‐destabilizing ability of 19 d ‐amino acid residues in an amphipathic α‐helical model peptide was evaluated by reversed‐phase HPLC and CD spectroscopy. l ‐Amino acid and d ‐amino acid residues show a wide range of helix‐destabilizing effects relative to Gly, as evidenced in melting temperatures (ΔT_m) ranging from ?8.5°C to 30.5°C for the l ‐amino acids and ?9.5°C to 9.0°C for the d ‐amino acids. Helix stereochemistry stability coefficients defined as the difference in T_m values for the l ‐ and d ‐amino acid substitutions [(ΔT_m′ = T_mL and T_mD)] ranging from 1°C to 34.5°C. HPLC retention times [Δt_R(X_L?X_D)] also had values ranging from ?0.52 to 7.31 min at pH 7.0. The helix‐destabilizing ability of a specific d ‐amino acid is highly dependent on its side‐chain, with no clear relationship to the helical propensity of its corresponding l ‐enantiomers. In both CD and reversed‐phase HPLC studies, d ‐amino acids with β‐branched side‐chains destabilize α‐helical structure to the greatest extent. A series of helix stability coefficients was subsequently determined, which should prove valuable both for protein structure‐activity studies and de novo design of novel biologically active peptides.     

Conformational studies of a potent Leu11, D-Trp12-containing lactam-bridged parathyroid hormone-related protein-derived antagonist

Human parathyroid hormone-related protein (PTHrP) is expressed in various tissues where it acts as an endocrine/paracrine factor involved in cellular growth, differentiation and development of fetal skeleton. As for parathyroid hormone (PTH), which is the hormone responsible for regulation of extracellular calcium homeostasis, the N-terminal 1-34 fragment can reproduce the full spectrum of calciotropic activities inherent in full-length PTH. Truncation of six amino acid residues from the N-terminus of both hormone sequences generates 7-34 fragments which act as weak antagonists. Although PTH(7-34) is a pure antagonist, PTHrP(7-34) acts as partial agonist against the receptor shared by both hormones, the PTH/PTHrP receptor. In the current study, we analyzed the conformation of [Leu¹¹,d -Trp¹²,Lys²⁶,Asp³⁰]PTHrP(7-34)NH₂ (hybrid-lactam) in a 1:1 mixture of H₂O/TFE-d₃ at pH 4 by circular dichroism, nuclear magnetic resonance and distance geometry calculations. This weak antagonist (K_b= 650 nm ) combines two modifications: Leu¹¹,d -Trp¹² (K_b= 5.1 nm ), reported to eliminate partial agonism and enhance potency, and Lys²⁶-Asp³⁰ lactamization (K_b= 31 nm ), aimed to stabilize the helical structure of the principal binding domain attributed to residues 25-34. The helical content in 30% trifluoroethanol is 88%, i.e., higher than the corresponding linear analog, and comprises the d -Trp¹²-Thr³³ segment. This hybrid lactam contains a rigid helical segment spanning the 14-18 sequence followed by a hinge motif around Arg^19-20, but the sequence 14-18 forms a stable helix. In all potent lactam-containing, PTHrP-derived agonists and antagonists studied so far, the dominant structural motif consists of two helical domains at the two ends of the sequence and of two hinge regions centered around Gly¹²-Lys¹³ and Arg¹⁹. The weakly active agonists and antagonists do not exhibit the “hinge” around position 19. These findings suggest that the presence and location of discrete hinge regions that connect the N- and C-terminal helices are essential for generating the bioactive conformation of ligands for the PTH/ PTHrP receptor.     

A New i,i + 3 Peptide Stapling System for α‐Helix Stabilization

We have previously shown that the incorporation of an 8‐atom all‐hydrocarbon ‘staple’ at positions i and i + 3 of a synthetic peptide results in substantial stabilization of the α‐helical conformation. As part of our ongoing effort to explore the scope and utility of all‐hydrocarbon stapling systems, we have investigated and report herein the properties of a new i, i + 3 stapling system that employs a 6‐carbon cross‐link.     

Structures of neuropeptide γ from goldfish and mammalian neuropeptide γ, as determined by 1H NMR spectroscopy

Abstract: Neuropeptide γ belongs to tachykinin families which have a common C‐terminal amino acid sequence (Phe‐X‐Leu‐Met‐NH₂) and which induce various biological responses including salivation, hypotension, and contraction of gastrointestinal, respiratory, and urinary smooth muscle. In the present study, we present the solution structures of neuropeptide γ (NPγ) from gold fish (G‐NPγ) and mammalian NPγ (M‐NPγ), as determined by nuclear magnetic resonance (NMR) spectroscopy in 50% trifluoroethanol (TFE)/water (1 : 1, v/v) solution and 200 mm sodium dodecyl sulfate (SDS) micelles. In aqueous TFE solution, G‐NPγ has a α‐helical conformation in the region of His¹²–Met²¹ and a short helix in the N‐terminal region, and has a β‐turn from Arg⁹ to Arg¹¹ in between. In aqueous TFE solution, M‐NPγ also has α‐helical conformations both in the C‐terminal region and the N‐terminal region and a β‐turn from His⁹ to Arg¹¹ in between. In SDS micelle, the structure of G‐NPγ contains a stable α‐helix from His¹² to Met²¹ and a β‐turn from Arg⁹ to Arg¹¹, while M‐NPγ has a short helix from Ser¹⁶ to Met²¹. The region from His¹² to Met²¹ corresponds to the amino acid sequence of neurokinin A. Neuropeptide γ may act as a precursor of neurokinin A and the post‐translational processing of this peptide involves the enzymatic attack of the basic β‐turn region from residue 9 to residue 11 in the middle. From our relaxation study, it could be suggested that in fish system G‐NPγ induces the biological actions corresponding to those of substance P in mammalian system. The structures of G‐NPγ and M‐NPγ contain α‐helical structures at the C‐terminus and this helix seems to promote the affinity for NK₁ and/or NK₂ receptor.     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: MOLECULAR DETERMINANTS OF PEPTIDE AND NON-PEPTIDE BINDING TO THE AT1 RECEPTOR

• 1 Several residues critically involved in AT₁ receptor ligand-binding and activation have now been identified based on mutational and biochemical studies.
• 2 Asp²⁸¹ and Lys¹⁹⁹ of the rat AT₁ receptor ion-pair with Arg² and the Phe³ α-COOH of angiotensin II (AngII), respectively, and the Asp²⁸¹/Arg² interaction is critical for full agonist activity.
• 3 Agonist activity of AngII also requires an interaction of the Phe² side chain with His²⁵⁶, which is achieved by docking of the α-COOH with Lys¹⁹⁹. Non-peptide agonists interact with Lys¹⁹⁹ and His²⁵⁶ in a similar fashion.
• 4 The crucial acid pharmacophores of AngII and the non-peptide antagonist, Iosartan, appear to occupy the same space within the receptor pocket. Binding of the tetrazole anion moiety of losartan involves multiple contacts, such as Lys¹⁹⁹ and His²⁵⁶. However, this interaction does not involve a conventional salt bridge, but rather an unusual lysine-aromatic interaction.
• 5 Asp¹ of AngII forms an ion-pair with His¹⁸³, which stabilizes the receptor-bound conformation of AngII but is not critical for receptor activation.
• 6 These interactions and the involvement of other residues in stabilizing the wild-type receptor conformation or in receptor/G-protein coupling are considered here.
• 7 Despite these insights, considerable effort is still needed to elucidate how ligand binding induces receptor activation, what determines the specificity of AT₁ receptor coupling to multiple G-proteins and the in vivo role of receptor down-regulation.

     

Studies on the conformational properties of CP‐1042−55, the hinge region of CP‐10, using circular dichroism and RP‐HPLC

Abstract: The conformational properties of CP‐10^42?55, a peptide corresponding to the hinge region of CP‐10, were investigated using circular dichroism spectroscopy and reverse‐phase high‐performance liquid chromatography (RP‐HPLC). The circular dichroism studies indicated that CP‐10^42?55 formed considerable secondary structure in the presence of hydrophobic solution environments including 50% acetonitrile, 50% trifluoroethanol and 200 mm sodium dodecyl sulfate, which comprised a mixture of α‐helix and β‐sheet. The effect of temperature on the conformation of CP‐10^42?55 was investigated between 5 and 40°C, with very small changes in the spectra being observed.RP‐HPLC was then used to investigate the effect of temperature on the conformation of CP‐10^42?55 in the presence of a hydrophobic surface. Using a C₁₈‐adsorbent, CP‐10^42?55 exhibited a conformational transition at 25°C, which was associated with an increase in the chromatographic contact area and the binding affinity of the peptide for the stationary phase. In addition, near‐planar bandbroadening behaviour indicated that conformational species interconverted with rapid rate constants compared with the chromatographic time scale. These results indicated that the conformational change at 25°C in theRP‐HPLC system most likely corresponds to the unfolding of an α‐helical and/or β‐sheet structure to an extended coil structure. Therefore, the strong chemotactic properties of this peptide may be attributed to its ability to form considerable secondary structure in the presence of a hydrophobic environment.     

Solution conformation of Leu27 hGRF(1-32)NH2 and its deamidation products by 2D NMR

The solution structure and helical content of a human growth hormone releasing factor analog, Leu²⁷ hGRF(1–32)NH₂ (hGRF), and its deamidation products Asp⁸ Leu²⁷ hGRF(l-32)NH₂ and isoAsp⁸ Leu²⁷ hGRF(1-32)NH₂, were determined by CD and 2D NMR. Chemical-shift assignments of ¹H NMR resonances were made from DQFCOSY, HOHAHA and NOESY spectra, and qualitative secondary structure was determined from NOESY spectra. 2D NMR studies in aqueous MeOH showed the Asn⁸, Asp⁸ and isoAsp⁸ hGRF analogs to have significant α-helical character. However, the β-linked isoAsp⁸ analog did not retain helical structure in the N-terminal region, most likely because of disruption of the hydrogen bonding pattern upon substitution of the extra methylene into the peptide backbone. The helical content, as determined by CD, was ~ 12% in 0% MeOH for all three peptides, and 77, 72 and 69% in 80% MeOH for the Asn⁸, Asp⁸ and isoAsp⁸ hGRF analogs, respectively. However, 2D NMR solution structure data indicated a decrease in helicity in the N-terminal region for the isoAsp⁸ analog when compared with the other two analogs. In the Asn⁸ and Asp⁸ hGRF analogs, the helix began at Asp³ or Ala⁴, while the isoAsp⁸ analog helix was disrupted until Arg. The higher helicity value for the Asn⁸ peptide over the isoAsp⁸ analog may be associated with reported biological activity, where the in vitro activity decreased from 100 to 4 and < 1% for Asn⁸, Asp⁸ and isoAsp⁸ hGRF, respectively.     

Relationship between the tertiary structures of mastoparan B and its analogs and their lytic activities studied by NMR spectroscopy

Abstract: Mastoparan B (MP‐B), an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis, is an amphiphilic α‐helical peptide. MP‐B possesses a variety of biological activities, such as mast cells degradation histamine release, erythrocyte lysis and inhibition of the growth of Gram‐positive and Gram‐negative bacteria. In order to study the relationship between the structure and the biological activity of MP‐B, we used four analogs by replacing amino acids with alanine. Tertiary structures of MP‐B and its analogs in 2,2,2‐trifluoroethanol (TFE)‐containing aqueous solution have been determined by NMR spectroscopy and molecular modeling. The results indicate that [Ala⁴]MP‐B and [Ala¹²]MP‐B with higher hydrophobicity adopt a higher content of amphiphilic helical structures, and have better antimicrobial and hemolytic activities than MP‐B. However, [Ala³]MP‐B and [Ala⁹]MP‐B with lower hydrophobicity have disordered structures. [Ala³]MP‐B and [Ala⁹]MP‐B have low antimicrobial activity and much less hemolytic activity relative to MP‐B. It is likely that tryptophan residue in MP‐B and appropriate hydrophobicity of MP‐B to induce α‐helical structure is essential for the antibacterial and hemolytic activity of MP‐B. This study can aid understanding of the structure–activity relationship of MP‐B and to design peptides to possess lytic activity.     

The cytoplasmic helix of cannabinoid receptor CB2, a conformational study by circular dichroism and 1H NMR spectroscopy in aqueous and membrane‐like environments

Abstract: The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous G protein‐coupled receptors (GPCRs) such as rhodopsin and the β‐adrenergic receptor exhibit unique structural and functional characteristics. Computer models also predict this structure for the cannabinoid CB2 receptor, another member of the GPCR superfamily. In our study, a peptide corresponding to helix 8 of the CB2 receptor was synthesized chemically and its secondary structure determined by circular dichroism (CD) and ¹H NMR spectroscopy. NMR and CD revealed an α‐helical structure in this region in both dodecylphosphocholine micelles and dimethylsulfoxide, in contrast to a random coil configuration found in aqueous solvent. This finding is in good agreement with other previous GPCR structural studies including X‐ray crystallography. By combining our finding with other studies, we further hypothesize that the amphipathic nature of helix 8 can play a significant role in the function and regulation of CB receptors as well as other GPCRs in general.     

Tryptophan‐containing peptide helices: interactions involving the indole side chain*

Abstract: Two designed peptide sequences containing Trp residues at positions i and i + 5 (Boc‐Leu‐Trp‐Val‐Ala‐Aib‐Leu‐Trp‐Val‐OMe, 1 ) as well as i and i + 6 (Boc‐Leu‐Trp‐Val‐Aib‐Ala‐Aib‐Leu‐Trp‐Val‐OMe, 2 ) containing one and two centrally positioned Aib residues, respectively, for helix nucleation, have been shown to form stable helices in chloroform solutions. Structures derived from nuclear magnetic resonance (NMR) data reveal six and seven intramolecularly hydrogen‐bonded NH groups in peptides 1 and 2 , respectively. The helical conformation of octapeptide 1 has also been established in the solid state by X‐ray diffraction. The crystal structure reveals an interesting packing motif in which helical columns are stabilized by side chain–backbone hydrogen bonding involving the indole N?1H of Trp(2) as donor, and an acceptor C=O group from Leu(6) of a neighboring molecule. Helical columns also associate laterally, and strong interactions are observed between the Trp(2) and Trp(7) residues on neighboring molecules. The edge‐to‐face aromatic interactions between the indoles suggest a potential C‐H…π interaction involving the Cζ3H of Trp(2). Concentration dependence of NMR chemical shifts provides evidence for peptide association in solution involving the Trp(2) N?1H protons, presumably in a manner similar to that observed in the crystal.     

(αMe)Nva: stereoselective syntheses and preferred conformations of selected model peptides

Abstract: Using different stereoselective chemical and chemoenzymatic approaches we synthesized the chiral, C^α‐methylated α‐amino acid l ‐(αMe)Nva with a short, linear side‐chain. A set of terminally protected model peptides to the pentamer level containing either (αMe)Nva or Nva in combination with Ala and/or Aib was prepared using solution methods and characterized fully. Two (αMe)Nva peptides were also synthesized using side‐chain hydrogenation of the corresponding C^α‐methyl, C^α‐allylglycine (Mag) peptides. A detailed solution and crystal‐state conformational analysis based on FT‐IR absorption, ¹H NMR and X‐ray diffraction techniques allowed us to define that: (i) (αMe)Nva is an effective β‐turn and 3₁₀‐helix former; and (ii) the relationship between (αMe)Nva chirality and the screw sense of the turn/helix formed is that typical of protein amino acids, i.e. l ‐(αMe)Nva induces the preferential formation of right‐handed folded structures. In more general terms, this study reinforced previous conclusions that peptides based on α‐amino acids with a C^α‐methyl substituent and a C^α‐linear alkyl substituent are characterized by a strong tendency to fold into turn and helical structures.     

Conformational mapping of the N‐terminal segment of surfactant protein B in lipid using 13C‐enhanced Fourier transform infrared spectroscopy

Abstract: Synthetic peptides based on the N‐terminal domain of human surfactant protein B (SP‐B_1?25; 25 amino acid residues; NH₂‐FPIPLPYCWLCRALIKRIQAMIPKG) retain important lung activities of the full‐length, 79‐residue protein. Here, we used physical techniques to examine the secondary conformation of SP‐B_1?25 in aqueous, lipid and structure‐promoting environments. Circular dichroism and conventional, ¹²C‐Fourier transform infrared (FTIR) spectroscopy each indicated a predominateα‐helical conformation for SP‐B_1?25 in phosphate‐buffered saline, liposomes of 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol and the structure‐promoting solvent hexafluoroisopropanol; FTIR spectra also showed significant β‐ and random conformations for peptide in these three environments. In further experiments designed to map secondary structure to specific residues, isotope‐enhanced FTIR spectroscopy was performed with 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol liposomes and a suite of SP‐B_1?25 peptides labeled with ¹³C‐carbonyl groups at either single or multiple sites. Combining these ¹³C‐enhanced FTIR results with energy minimizations and molecular simulations indicated the following model for SP‐B_1?25 in 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol: β‐sheet (residues 1–6), α‐helix (residues 8–22) and random (residues 23–25) conformations. Analogous structural motifs are observed in the corresponding homologous N‐terminal regions of several proteins that also share the ‘saposin‐like’ (i.e. 5‐helix bundle) folding pattern of full‐length, human SP‐B. In future studies, ¹³C‐enhanced FTIR spectroscopy and energy minimizations may be of general use in defining backbone conformations at amino acid resolution, particularly for peptides or proteins in membrane
environments.     

Structural features of linear,homo‐Aib‐based peptides in solution: a spectroscopic and molecular mechanics investigation

Abstract: In continuation of our studies on the determination of the structural features of functionalized peptides in solution by combining time‐resolved fluorescence data and molecular mechanics results, the conformational properties of a series of linear, homo‐Aib peptides in methanol (a structure‐supporting solvent) were investigated. These compounds have the general formula P(Aib)_nN, where Aib is α‐aminoisobutyric acid, N is naphthalene and P is the monomethylated protoporphyrin IX, the two latter chromophores being covalently attached to the peptide C‐ and N‐termini, respectively, while n = 3, 6 and 9. According to ¹H NMR and IR spectra, the peptides investigated largely populate a 3₁₀‐helical structure in CDCl₃, which is also a structure‐supporting solvent. Both steady‐state and time‐resolved fluorescence measurements show a strong quenching of the N emission that parallels an increase of the P fluorescence intensity, suggesting the occurrence of long‐range energy transfer from ¹N* to ground‐state P. Comparison of quenching efficiencies and lifetime pre‐exponents with those obtained theoretically from the deepest energy minimum conformers is very satisfactory. The computed structures, built up by partially taking into account the solvent medium, exhibit a rigid, highly compact arrangement, owing to both the 3₁₀‐helix conformation of the backbone chain and the very few peptide‐to‐chromophore covalent linkages. As a result, only one or two stable conformations for each peptide were theoretically found, in full agreement with the time‐resolved fluorescence data. Orientational effects between the probes must be taken into account for a correct interpretation of the fluorescence decay results, which implies that interconversion among conformational substates of the N linkages is slower than 10 ns, corresponding to the upper limit of the energy transfer
characteristic time.     

Circular dichroism and Fourier transform infrared spectroscopic studies on self‐assembly of tetrapeptide derivative in solution and solvated film

Abstract: Aggregation of the hydrophobic peptide derivative Boc‐Ala‐Ile‐Ile‐Gly‐OMe ( 1 ) was examined in methanol solution and in solvated film states. Formation of the peptide by self‐assembly was evidenced using fluorescence [Mg salt of 8‐anilino‐naphthalenesulfonic acid (ANS) as an external probe] and circular dichroism (CD) spectroscopic techniques. In solution, peptide 1 formed as a stable aggregate at a concentration around 3 × 10^?4m . The peptide gelled into a thin film for which we carried out CD and Fourier transform infrared (FTIR) measurements. Our spectroscopic study on peptide films at differing methanol concentrations indicates that the helical content of the peptide decreases with decreasing methanol concentration in solvated films. However, by reducing the methanol concentration we were able to observe a conformational transition from a predominantly helical turn to a β‐sheet structure via a random coil conformation. Our study focused on the aggregation of the α‐helical turn‐forming peptide derivative, which shows conformational transition on changing solvent concentration in the film form.     

Structure–activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin

Abstract: Peptides derived from the third α‐helix of the homeodomain (residues 43–58; Penetratin) of Antennapedia, a Drosophila homeoprotein, were prepared by simultaneous multiple synthesis. Sets of N‐ and C‐terminally truncated peptides, as well as a series of alanine substitution analogues, were studied. Cell penetration assays using human cell cultures with these peptides revealed that the C‐terminal segment ⁵²Arg‐Arg‐Met‐Lys‐Trp‐Lys‐Lys⁵⁸ of the parent sequence was necessary and sufficient for efficient cell membrane translocation. Individual Ala substitutions of the peptide’s basic residues led to markedly decreased cell internalization ability, whereas replacement of hydrophobic residues was tolerated surprisingly well. Subcellular localization was seen to be affected by substitutions, with analogues being addressed preferentially to the cytosol or to the nucleus. Conformational constriction of the Penetratin sequence through placement and oxidation of flanking cysteine residues afforded a cyclic disulfide peptide which had lost most of its membrane translocation capacity.     

(αMe)Aun: a highly lipophilic,chiral, Cα‐tetrasubstituted α‐amino acid. Incorporation into model peptides and preferred conformation

Abstract: Using a chemo‐enzymatic approach we prepared the highly lipophilic, chiral, C^α‐methylated α‐amino acid (αMe)Aun. Two series of terminally protected model peptides containing either d ‐(αMe)Aun in combination with Aib or l ‐(αMe)Aun in combination with Gly were synthesized using solution methods and fully characterized. A detailed solution conformational analysis, based on FT‐IR absorption, ¹H NMR and CD techniques, allowed us to determine the preferred conformation of this amino acid and the relationship between chirality at its α‐carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that d ‐(αMe)Aun favors the formation of the left‐handed 3₁₀‐helical conformation.     

Effects of Na2SO4 on hydrophobic and electrostatic interactions between amphipathic α‐helices

Abstract: The effects of 2 molal Na₂SO₄ at neutral pH on hydrophobic and electrostatic interactions between amphipathic α‐helices were investigated by circular dichroism spectroscopy. The amphipathic peptides that were studied included LEK (acetyl‐ L EE L KKK L EE L KKK L EE L ‐NH₂) and LEE (acetyl‐ L EE L EEE L EE L EEE L EE L ‐NH₂). In phosphate buffer at neutral pH, only LEK adopted a predominantly α‐helical conformation, attributable to glu^––lys⁺ interactions where a major contribution is evidently a hydrogen bond (Biochemistry 32 : 9668–9676). Despite the presence of lys⁺ in the e and g′ positions of the abcdefg heptad repeat, LEK exhibited mean‐residue ellipticities at 222 nm ([θ]₂₂₂) which were dependent on peptide concentration, indicating the presence of a coiled coil. In the presence of 2 molal Na₂SO₄ at 25–75°C, the helical content of LEK increased, with the greatest increase observed at 75°C. The value of the ellipticity ratio R ([θ]₂₂₂/[θ]₂₀₈) of LEK in 2 molal Na₂SO₄ also increased, indicating a stronger interhelical association. At 50°C and 75°C, LEK remained predominantly α‐helical. In phosphate buffer at neutral pH, LEE was mainly random coil. In the presence of 2 molal Na₂SO₄, however, the peptide formed α‐helices that associated to form a coiled coil. At 50°C and 75°C, LEE became predominantly random coil but the remaining α‐helices were still associating. These results are consistent with the strengthening of interhelical hydrophobic interactions and the absence of screening of helix‐stabilizing and helix‐destabilizing electrostatic interactions in amphipathic α‐helices by Na₂SO₄.