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Circular dichroism studies of ampullosporin‐A analogues

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  Circular dichroism studies of ampullosporin‐A analogues
   Journal of Peptide Science  J. Peptide Sci.  9 : 714–728 (2003)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/psc.459 Circular Dichroism Studies of Ampullosporin-A Analogues ‡ HOAI-HUONG NGUYEN, a DIANA IMHOF, a MATTHIAS KRONEN, b UDO GR¨AFE b § and SIEGMUND REISSMANN a * a Institut f¨ur Biochemie und Biophysik, Friedrich-Schiller-Universit¨at Jena, Philosophenweg 12, D-07743Jena, Germany b Hans-Kn¨oll-Institut f¨ur Naturstoffforschung, Beutenbergstraße 11, D-07745 Jena, Germany Received 3 July 2002  Accepted 16 December 2002   Abstract: Ampullosporin A (AmpA), a 15mer peptaibol containing seven Aib residues is able to inducepigmentation on  Phoma destructiva   and hypothermia in mice, as well as to exhibit a neuroleptic effect. A circular dichroism study of ampullosporin A and its analogues was carried out in organic solvents with different polarities and detergent micelles to determine the relationship between their conformationalflexibility and biological activities. The analogues were obtained by modifying the  N  - and  C  -termini of ampullosporin A. Furthermore, Gln and Leu were systematically substituted by Ala and Aib residues werereplaced by Ala and/or Ac 6 c. To estimate the helicity of the analogues, the CD spectrum of AmpA recordedin acetonitrile was correlated to its crystal structure. All analogues displayed similar CD curve shapes inorganic solvents with the ratio between two negative band intensities  R   = [ θ  ] n  − π ∗ / [ θ  ] π − π ∗  <  1. In acetonitrile,most of theanalogues adopted a 70%–85% helical structure, whichwashigherthan the average of 40%–60%obtained in TFE. In detergent micelles, the analogues were distinguishable by their CD profiles. For most of the biologically active analogues, the CD spectra in detergent micelles were characterized by a   R   ratio  >  1and increased helicity compared with those recorded in TFE, suggesting that the interaction of the peptides with the membrane and peptide association was necessary for their hypothermic effect. Copyright   ©  2003European Peptide Society and John Wiley & Sons, Ltd.Keywords: peptaibol; conformational study; circular dichroism  Abbreviations: Abbreviations used for amino acids follow theRecommendations of the IUPAC-IUB Biochemical Nomenclature( Eur. J. Biochem.  1984;  138 : 9–37). The following additionalabbreviations are used throughout the text: Ac 6 c, 1-amino-1-cyclohexane carboxylic acid; AmpA (B, C, D), ampullosporin A (B,C, D); CD, circular dichroism; CMC, critical micellar concentration;DPC, dodecyl phosphocholine; EtOH, ethanol; HFIP, 1,1,1,3,3,3-hexafluoroisopropanol; iPrOH, isopropanol; MeOH, methanol;MeCN, acetonitrile; MRE, mean residue ellipticity; NMR, nuclear magnetic resonance; Oic, octahydro-1H-indole-2-carboxylic acid;SDS, sodium dodecylsulphate; TFE, 2,2,2-trifluoroethanol; Tic,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.*Correspondence to: Professor Siegmund Reissmann, Institut f¨ur Biochemie und Biophysik, Friedrich-Schiller-Universit ¨at Jena,Philosophenweg 12, D-07743 Jena, Germany;e-mail: [email protected]‡Presented at Peptaibols, October 2002, Jena, Germany.§Deceased.Contract/grant sponsor: Deutsche Forschungsgemeinschaft; Con-tract/grant number: 853/7-1. INTRODUCTION  A number of natural ampullosporin analogues:ampullosporin A (AmpA), ampullosporin B (AmpB),ampullosporin C (AmpC) and ampullosporin D(AmpD) have been isolated from the fungus  Sepe- donium ampullosporum   [1,2]. They belong to thepeptaibol class of natural products, character-ized by the presence of an acetylated  N  -terminus,a   C  -terminal amino alcohol and a high propor-tion of   α -aminoisobutyric acid (Aib). Peptaibols were reported to exhibit membrane permeabiliz-ing activity [3–5]. For instance, alamethicin [5]and chrysospermin [6] can form ion channels, whereas antiamoebin [7] acts as an ion carrier and trichogin [8] induces the leakage of aqueouscontent from liposomes. Ampullosporins, strongly  Copyright   ©  2003 European Peptide Society and John Wiley & Sons, Ltd.   AMPULLOSPORIN A ANALOGUES  715 hydrophobicand devoid of charges,are also believedto have a site of action in the membrane. The Trp residue at the  N  -terminus may interact withthe bilayer interface as a membrane anchor [9,10]. The membrane conductance as a function of nat-ural ampullosporin concentration showed the for-mation of ion conducting pores in the artificial bilayer [11,12]. The conformation of AmpA, solved by x-ray diffraction, is a largely regular   α -helix starting fromthe acetylated  N  -terminus and a   β -turn at the C  -terminus [13]. In similarity to alamethicin [14]and chrysospermin [6], AmpA helices possess a hydrophilic face formed by the polar side chainsof Gln 7 , Gln 11 , and non hydrogen bonding carbonyloxygens of Aib 10 and Gln 11 , while the hydrophobicface is formed by the bulky side chains of  Trp 1 ,  Leu 5 ,  Leu 12 and Leu 15 ol. The length of AmpA in the crystals is only 23 ˚ A, much shorter thanthat of antiamoebin (28.3 ˚ A), a 16mer peptaibol[7]. Compared with the core thickness of the bilayer, which varies from 19.5 ˚ A to 29 ˚ A [5], a single molecule of AmpA hardly spans the standard bilayer even when it produces a membrane thinningeffect. On the other hand, the crystal packingof AmpA shows its ability to self-associate via hydrogen bonds in a head-to-tail fashion and inantiparallel fashion due to polar side chains andthe non hydrogen bonding carbonyl oxygen of Aib 10 [13]. If this hydrogen bonding can also occur inthe membrane, the pore forming activity of AmpA might be explained according to the barrel-stavemodel by the flip-flop mechanism [15]. However,in contrast to alamethicin, AmpA displayed only a  weak antimicrobial effect [1]. The more interesting biologicalactivityof AmpA andits naturalanaloguesisrelatedtotheirabilitytoinducepigmentformationin the fungus  Phoma destructiva  , as do severalcommercial nonpeptide antipsychotics haloperidol,chlorpromazine, clozapine and sertindole [1,2]. Inaddition, natural ampullosporins can also provokehypothermia and inhibit locomotor activity inmice [1,2]. Specific behavioural tests confirmedthe assumption of an atypical neuroleptic activity of AmpA [16]. Whereas the biological activities of peptaibols,such as antimicrobial, antimalarial and haemolyticactivities, stimulation of catecholamine secretion,uncouplers of mitochondrial oxidative phosphoryla-tion [3,4] etc. were reported to be generated fromtheir membrane activities, the action mechanismsof the biological activities of ampullosporins remainunknown. To elucidate the relationship betweenthe conformation and biological activities of ampul-losporins, a set of AmpA analogues has been syn-thesized by solid phase peptide synthesis and their  biological activities have been tested [17]. The ana-logues are different in their   C  - and  N  -termini andalso in the number of Aib residues. Some analoguesretain as high pigment induction activity and asstrong hypothermic effect as AmpA whereas othersexhibit strong pigment induction activity together  with a significantly reduced hypothermic response. Analogues without aromatic residues or 14mer ana-logues are devoid of any biological activity. Further-more, the acetylated  N  -terminus and hydroxylatedor amidated  C  -terminus seem to play an important role in the hypothermic activity, too. The replace-ment of Aib residues by Ala or Ac 6 c (see Abbrevia-tions) at different sequence positions affected like- wise the biological activities. To check the effect of side chains on biological activities of ampullosporinanalogues, Gln and Leu residues were individu-ally substituted by Ala. Whereas replacement of every Gln by Ala did not affect the biological activ-ities, the lack of a Leu side chain led to a loss of pigment induction and to a reduction of hypother-mic response.In the present paper, we report the results of a conformational study of natural and syntheticampullosporin analogues, using circular dichroism.Circular dichroism is a valuable tool for estimatingthe secondary structure of proteins and peptidesin solution, especially for analysing their conforma-tional change in different environments. To examinethe conformation of the peptide in the membranes,detergent micelles SDS and DPC were used, whichare considered as membrane mimetic solvents withhydrophobicand hydrophilic interface and which donot distort the spectra from differential light scatter-ing and differential absorption flattening [18]. Thereare many methods for secondary structure analysisfrom CD spectra. However, the accuracy of the esti-mationdependsstronglyonthereferenceproteinset [19]. Since AmpA and most of its analogues containnearly 50% of achiral Aib residues, the use of ref-erences from proteins and polypeptides containingusual amino acids is not appropriate. In this study,the helical content of the analogues was determinedat a single wavelength of about 222 nm with refer-ence data taken from the crystal structure of AmpA. MATERIALS AND METHODS Natural AmpB and AmpD were isolated from Sepedonium ampullosporum   HKI-0053 [2]. AmpA, Copyright   ©  2003 European Peptide Society and John Wiley & Sons, Ltd.  J. Peptide Sci.  9 : 714–728 (2003)   AMPULLOSPORIN A ANALOGUES  717  AmpC and other analogues were synthesized by solid phase method, using the automated pep-tide synthesizer ECOSYN P (Eppendorf, Biotronik)(Table 1). Details of the synthesis, cleavage, purifi-cation and characterization of the peptides werereported elsewhere [17]. MeOH, EtOH, iPrOH, TFE,HFIP, MeCN (for UV-spectroscopy or GC) werepurchased from Fluka (Steinheim, Germany), andFisher Chemicals (Loughborough, UK), SDS andDPC were from Sigma Aldrich Chemie GmbH (Stein-heim, Germany) and Avantipolar Lipids (Alabaster,USA),respectively.CDspectrawererecordedusinga  Jasco model 720 spectropolarimeter (Umstadt, Ger-many). The optical rotation was calibrated usingd-camphorsulphonic acid at 290.5 nm [20]. Unlessotherwise stated, measurements of three scans wererecorded using rectangular quartz cells of 0.1 cmpathlength, 2 nm bandwidth, 20 nm/min scanspeed, 8 s time constant, and at 22 ° C. The back-ground spectra were recorded in the same solvent  without peptide and then subtracted. The spectra reported here are the average of three experiments.Except for the CD spectra recorded in water, other spectra were obtained with samples prepared fromstock solutions of 1 m M  peptide in TFE. TFE con-centration in samples for CD measurements never exceeded 10% and equivalent concentrations of TFE were added to control samples. In samples con-taining detergents,phosphatebuffer saline pH = 7 . 4 was added at 10 m M , DPC and SDS concentrations were 20 and 50 m M , respectively.Peptide stock solu-tion concentrations were determined by measuringthe absorbance of Trp at 280 nm in 6  M  guanidinehydrochloride,10 m M  phosphatebuffer saline (PBS),pH = 7 . 4, using  ε 280 nm  = 5690 M − 1 cm − 1 [20].Ellipticity at about 222 nm ( n  - π ∗ transition) isassumed to be linearly related to the mean helix content f  H , which can be calculated from the Lifson-Roig based helix-coil model [21]:f  H  = ( [ θ  ] 222 − [ θ  ] C )/( [ θ  H ] − [ θ  ] C ), ( 1 )  where [ θ  ] 222 , [ θ  ] C  and [ θ  ] H  are mean residue elliptic-ities at about 222 nm of the calculated spectrum,random coil and complete helix, respectively. Thelatter two quantitiesare temperaturedependentandcan be determined as follows [21]:[ θ  ] C  = 2220 − 53 T  , ( 2a  ) [ θ  ] H  = ( − 44000 + 250 T  )( 1–3 / Nr  ), ( 2b )  where  T   is temperature in  ° C, and Nr is the chainlength in residues. At 22 ° C, [ θ  ] C  = 1054 deg cm 2 dmol − 1 and[ θ  ] H  =− 30800 deg cm 2 dmol − 1 for15mer peptides containing usual amino acids. The mean helix content f  H  can also be obtained by averaging the hydrogen bond probabilities of allresidues which can hydrogen bond:f  H  = n H /( Nr  − 3 ), ( 3 )  where n H  is the average number of helical hydrogen bonds and (Nr-3) is the number of hydrogen bondspossible in a peptaibol. RESULTS AND DISCUSSION CD Spectra of AmpA Comparison of CD spectra of AmpA in water and different organic solvents  .  To correlate the CDspectraof AmpA with its crystalstructure(Figure 1),CD spectra of AmpA were recorded in a seriesof organic solvents, including TFE, HFIP, MeOH,EtOH, iPrOH, as well as MeCN, from which thecrystals were obtained [13]. Alcohols, particularly  TFE and HFIP, have been reported to effectively induce helical structure over other alcohols due tohydrophobic interactions of the solvent moleculesand hydrophobic groups of the peptides [22,23].However, according to Reichardt’s overall solvationability concept (termed also ‘polarity’, which ismeasured using the Dimroth-Reichardt’s parameter E  T  ( 30 )  [24]), HFIP and TFE as well as water arestrongly polar and MeCN expresses only mediumpolarity. In alcohols, AmpA is soluble, while inMeCN, it is totally soluble only with the addition of approximately 40% water. However, AmpA crystals wereobtainedfromMeCNwiththepresenceoftracesof water. To obtain a concentration independent CDspectrum of AmpA in an MeCN rich environment,10% TFE was used as a cosolvent. The CD spectra of AmpA in different solvents aregiven in Figure 2. With seven Aib, three Leu andone Trp residues, AmpA is very hydrophobic, andhence, its CD spectrum in water is concentrationdependent. At a concentration  > 40  µ M , one negativepeak at about 205 nm and one positive peak near 194 nm were recorded, while at a lower concentration, the negative peak shifted to 203 nmand the positive peak became negative, suggesting a high random coil content at the lower concentration(data not shown). The CD spectra of AmpA in MeOH, EtOH, iPrOHand MeCN possessing two pronounced negative Copyright   ©  2003 European Peptide Society and John Wiley & Sons, Ltd.  J. Peptide Sci.  9 : 714–728 (2003)  718  NGUYEN  ET AL  . Figure 1 Crystal structure of AmpA, dot lines: hydro-gen bonds. minima at 223 nm and 208 nm and a positivemaximum near194 nm aretypicalfor   α -helix.Thesespectra were comparable not only in curve shapes, but also in band magnitudes. Interestingly, the CDspectrum of AmpA in TFE resembled the waveformof those in the above described organic solvents with the following differences: the  π - π ∗   transitionshifted to 206 nm and the intensities of all peaks were significantly reduced. Furthermore, the CDspectrum in HFIP showed one negative peak at 204 nm and a very weak positive peak at about 194 nm. It is likely that the random coil content increased when TFE and especially HFIP were usedas solvents. Besides, AmpA is rich in Aib, whichhave been reported to promote the onset of 3 10 -helix due to steric interactions involving the  gem  -methylgroups linked to the  α -carbon [25]. In Aib containingpeptides, the 3 10 -geometry is energetically favouredover the  α -geometry. Therefore, Aib containing Figure 2 CD spectra of AmpA in water, HFIP, TFE, iPrOH,MeCN, EtOH and MeOH from the top to bottom at 222 nm,peptide concentrations were 86  µ M . peptides adopt pure 3 10 -, pure  α -, or mixed 3 10 -/ α -helices depending on their length and the relative Aib content. In peptides of eight or more residues, a pure  α -helix was observed if the Aib content did not exceed 50% [25]. In the crystal structure of AmpA,an  α -helical segment from CO of acetyl group toNH of Aib 9  was found (Figure 1). However, several weak hydrogen bonds of type  i  ,  i  + 3 were present concomitantly with  α -helical hydrogen bonds in themiddle of the molecule [13]. The theoretical CDspectrum of 3 10 -helix calculated by Manning and Woody[26]agreedwellwithToniolo’smeasurements[27], whichshowedthat the right-handed3 10 -helicalpeptide displayed a negative CD band at 207 nmaccompanied by a shoulder centred near 222 nm,and the ratio  R   = [ θ  ] 222 / [ θ  ] 207  of 0.4. Analysing CDspectra of AmpA in organic solvents, it was foundthat the  R   values are 0 . 83 ± 0 . 03 in MeOH, EtOH,iPrOH and MeCN, but only 0.73 and 0.36 in TFEand HFIP, respectively. According to [26,27], usingthe  R   value to diagnose the presence of a 3 10 -helix,one can say that in TFE, AmpA adopted a mixture of  α - and 3 10 -helix and in HFIP, the 3 10 -helical content may be higher. Besides the 3 10 -helix, other typesof   β -turn may occur, for example, a   β -turn type I.Examining numerous  β -turn types [28], it is worth being interested only in  β -turn types I and III for  AmpA analogues. On the basis of the studies on theCD spectra of   β -turn by Woody and Chang [29,30],it was assumed that in TFE and HFIP, the presenceof   β -turns (including 3 10 -helix) in different contents Copyright   ©  2003 European Peptide Society and John Wiley & Sons, Ltd.  J. Peptide Sci.  9 : 714–728 (2003)
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