Conformational studies of the cyclic l, d -lipopeptide surfactin by Fourier transform infrared spectroscopy

SPECTROCHIMICA ACTA PART A

ELSEVIER

Spectrochimica Acta Part A 53 (1997) 623 635

Conformational studies of the cyclic L,D-lipopeptide surfactin by Fourier transform infrared spectroscopy G. Ferr6, F. Besson, R. Buchet * Laboratoire de Physico-Chimie Biologique, UPRESA-CNRS 5013, Universit~ Claude Bernard-Lyon 1. Batiment 303, 43 Boulevard du 11 novembre 1918, F-69622 Villeurbanne, Cedex, France

Received 20 May 1996; revised 2 July 1996: accepted 12 July 1996

Abstract

The infrared spectra of acidic surfactin, its mono- and di-methylester derivatives were measured in dry state, 2H20, dimethylsulfoxide and trifluoroethanol. The IR spectra of the lipopeptides show two component bands at 1737 1742 and 1718 cm 1 indicating the presence of a mixture of non hydrogen-bonded and hydrogen-bonded C - O groups of the lactone ring. These bands overlapped with the C ~ ) groups of the Asp and Glu residues of protonated surfactin. Changes in relative intensities of the 1737 1742 and 1718 cm l bands were assigned to modifications of population of hydrogen-bonded carbonyl groups, involving solvent molecules and/or other groups of the lipopeptides. The IR spectra of surfactin and its derivatives display in the amide I region, at least, three component bands located at 1672-1681, 1656-1659 and 1641-1649 cm-1. The position and the magnitude of each component band depend on the natures of the solvent and of the lipopeptide. The 1641 1649 cm t band was indicative of a hydrogen-bonded C-O group as probed by temperature variation and was tentatively assigned to C ~ ) group involved in fl-turn. Slight spectral differences between surfactin and its derivatives may indicate small structural distorsion of the peptide backbone. We propose that the small structural differences between surfactin and its derivatives, observed on their respective IR spectra, could be related to their different biological properties. ~ 1997 Elsevier Science B.V. Keywords: Surfactin: Fourier transform infrared spectroscopy: Conformation; LD-lipopeptide; Organic solvent;

fl-turns

1. Introduction

Surfactin is a l i p o p e p t i d e p r o d u c e d by B a c i l l u s It is c o n s t i t u t e d by a L , o - h e p t a p e p t i d e m o i e t y cycled in a lactone ring by a f l - h y d r o x y

subtilis.

* Corresponding author. Tel.: + 33 4 72431320: fax: + 33 4 72431543: e-mail: [email protected]

fatty acid (Fig. 1) [l 3]. Recently several isoforms o f surfactin, differing by the p e p t i d e sequence, were identified [4-8]. Surfactin exhibited biological effects such as a n t i - H I V [8] a n d h a e m o l y t i c [9] activities, a n t i t u m o r activities against Ehrlich ascites c a r c i n o m a cells [10], inhibition o f fibrin clot f o r m a t i o n [11] a n d o f A M P c p h o s p h o d i esterase [12]. T h e s u r f a c t a n t a n d p o r e f o r m i n g p r o p e r t i e s o f surfactin were investigated by using n a t u r a l a n d m o d e l m e m b r a n e s [13-19]. F u r t h e r -

1386-1425/97 $17.00 © 1997 Elsevier Science B.V. All rights reserved. P11S1 386-1425(96)01787-4

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G. FerrO et al. "Spectrochimica Acta Part A 53 (1997) 623-635

more, a two-dimensional ~H-NMR study combined with molecular modeling proposed two possible conformations for surfactin [20]. Both structures were characterized by a 'horse saddle' topology for ring atoms on which were attached the two polar glutamic and aspartic acid side chains on the opposite direction to the aliphatic chain of the/?-hydroxy fatty acid. One conformation, S1, included a fl-turn with single hydrogen bond (between the NH of Leu2 and the C-O of Asp5) while the other, $2, contained three hydrogen bonds (between the NH of Leu7 and the C-~) of Asp5, between the NH of Val4 and the C--O of Leu2 and between the NH of Leu6 and the C--O of the fl-hydroxy fatty acid). It was proposed that polar groups of glutamic and aspartic acids could be involved in a chelating action with divalent ions [20]. The methylation of the carboxylic residues of surfactin affected its chelating [12], haemolytic and surfactant [21] properties. The aim of this study was to gain more insight into the effects of methylation on the structure of surfactin, for a better understanding of the biological activities of surfactin. In particular, the eventual presence of a fl-turn and of hydrogen bonds involving carbonyl groups of the peptide backbone and the carboxylic side chain residues in surfactin and its methylester derivatives could be monitored by Fourier transform infrared (FTIR) spectroscopy. In this respect, FTIR spectroscopy is a powerful tool for identifying the conformation of proteins, polypeptides and membrane proteins [22-33]. Vankatachalam [34] was the first to characterize three types of fl-turns in tetrapeptides with L-amino acid residues where the peptide chain folds back on itself by forming a hydrogen bond between the C=O group of the ith amino acid residue and the NH of the (i + 3)th residue. Many types of fl-turns, differing by their dihedral angles, were further identified in proteins and polypeptides [35,36]. In the case of L,D-peptides, similar structures, where the peptide chain folds back on itself, were theoretically characterized by the length, angle and energy of their hydrogen bonds and were compared with experimental data [37]. Although the dihedral angles of /?-turns in such L,D-peptides are different from those of fl-turns in L-peptides, we used the termi-

nology of fl-turn (indicating a hydrogen bond between the C--O group of the ith amino acid residue and the NH of the ( i + 3)th residue) for surfactin as other authors in the case of cyclo (L-Ala D-Ala-amino caproyl) peptide with a type II fl-turn [23], of gramicidin S with a type II' /?-turn [38] and iturin with three fl-turns [39]. The FTIR spectra of the L,D-cyclopeptides containing /?-turns [23,36,38-40] formed the rationale basis for the interpretation of the FTIR spectra of surfactin and its derivatives. The fl-turns were also identified by using organic solvents since the exact position of the bands related to fl-turns depended on the solvent nature in the infrared spectra of L-synthetic cyclic peptides [41,42] or L,D-peptides such as gramicidin S [38], iturin [39] or cyclosporin [40].

2. E x p e r i m e n t a l

2.1. M a t e r i a l s

Deuterium oxide (99.9%) was purchased from Merck (Darmstadt, Germany). The acidic surfactin was purified from B. s u b t i l i s cultures as described in [6]. Since several analogs of surfactin [4-8] could be extracted from this type of cultures, the amino acid composition of the purified surfactin was checked by using a modified version ,c,,,e ('OOH i

NH CH CO - NH-CH-CO

/

I. Glu

CO

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L L~'u 7

D Lcu 6

L Asp 5

f

X CO-CH-NHI CH 2 CH I (CH~) 2

CO-CH-NH I CH 2 CH I

-

CO-CH-NH CH 2 COOH

(CH312

Fig. 1. Primarystructure of surfactin. The amino acid numbers are related to their position in the heptapeptide moiety of surfactin. The thick types indicates the Ci4-Cl7 fl-hydroxy fatty acid.

G. Ferr~~ et al.

Spectrochimica Acta Part A 53 (1997) 623 635

of the method described by Nimura and Kinoshita [43]. The gradient elution conditions were modified to optimize the analysis of the enantiomers. The amino acid titration gave the following molecular ratios, Asp: 1, Glu: 1, Val: 1 and Leu: 4. The surfactin diester (corresponding to the methylation of both the 7-COOH of Glu and the fl-COOH of Asp) was obtained as described in [12] and the surfactin monester (corresponding to the methylation of only the 7-COOH of Glu) was synthetized according to [21]. The purity of surfactin was tested by thin-layer chromatography on silica gel 60 in chloroform-methanol-water (65/ 25//4, v/v/v) and the purities of the surfactin esters by thin-layer chromatography on silica gel 60 in chloroform-methanol-30% NHaOH (13/5/1, v/v/ v). The impurities did not exceeded 5%. 2.2. Infrared spectroscopy Infrared spectra were recorded at 20°C (except when it was specified) by the mean of a Nicolet 510 M F T I R spectrometer. One hundred and twenty eight scans at a 4 cm ~ resolution were taken, coadded and Fourier transformed for each sample. During the data acquisition, the spectrometer was continuously purged with dry air. The solvent spectrum was deduced from the sample spectrum taken under the same conditions. Each infrared spectrum is representative of at least three independent measurements. In some cases, the final infrared spectrum was fitted by the mean of a curve fitting program using the Lorentzian band shape components. The unknown wavenumber position and the number of component bands were estimated by using the second derivative spectrum. Although this method does not give necessarily the correct solution, it constitutes the best estimate, since it was based on a reproductible and unambiguous analysis. To obtain the best fit, the wavenumber position of each component band, determined with the second derivative spectrum, was fixed while their respective width and height were allowed to be varied. The about 1 cm-diameter film of surfactin was obtained by spreading 50 ~tl of a c h l o r o f o r m methanol (2/1, v/v) solution of surfactin (4 nag ml ~) on a CaF 2 window and by drying. Under

625

these conditions, the infrared absorbance of the 1650 cm ~ band (the most intense) was about 0.1. Since surfactin and its derivatives were poorly water-soluble, 50 ~tl of 2H20 (or 50 IA of a 10 mM Tris-HC1 buffer in 2H20, pZH 8.15) were added directly on the surfactin film. The concentration of surfactin and its derivatives was 10 mg m l - 1 in trifluoroethanol (TFE) and in dimethylsulfoxide (DMSO). Fifty microliter aliquot was taken and filled in a 50 gm pathlength CaF2 cell.

3. Results 3.1. FTIR spectra o f sutfactin, its monoester and diester derivatives in solid state The methylation of surfactin can be easily visualized in the 1700-1800 cm 1 region, corresponding to the C=O stretching mode of carboxylic acids and esters (Fig. 2). The F T I R spectrum of surfactin (Fig. 2A) shows two component bands of similar absorbances, located at 1737 and 1718 cm t. The 1718 c m ~ component band corresponded at least partly to the carboxylic groups of aspartyl and glutamyl residues of protonated surfactin. The esterification of these carboxylic groups produced a slight decrease in intensity of the 1718 cm ~ component band and a concomitant increase in intensity of the high wavenumber component band at 1737-1742 cm ~, reflecting the conversion of - C O O H groups to - C O O C H 3 groups in the monoester (Fig. 2B) and in the diester surfactins (Fig. 2C). The C=O stretching mode of the non-hydrogen bonded of the lactone ring contributes to the 1737 1742 cm ~ band of the mono- and the di-ester since the 1737-1742 cm ~ band was observed in the surfactin spectrum (Fig. 2A). The remaining component band centred at 1718 cm 1 on the diester spectrum (Fig. 2C) was assigned to the C=O stretching mode of the hydrogen-bonded lactone ring. The infrared spectrum of surfactin in the amide I region (Fig. 2A, solid line) showed a main band located at 1648 cm 1 and a shoulder centred at 1677 cm ~ The main band shifted from 1648 (Fig. 2A) to 1650 cm ~ for the monoester (Fig. 2B, solid line) and to 1654 cm 1 for the diester

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G. Ferr(" et al. ,, Spectrochimica Acta Part A 53 (1997) 623 635

3.2. FTIR spectra of surJactin, its monoester and diester derivatives in 2H20

|

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Fig. 2. FTIR spectra of surfactin (A), its monoester (B) and diester (C) derivatives in solid state (solid lines) and their respective second derivative spectra (dashed lines).

(Fig. 2C, solid line). The wavenumber positions (1681, 1656-1657 and 1642-1643 cm -1) and the number (three) of the amide I component bands were estimated by using the second derivative spectra (Fig. 2, dashed lines). The result of the best fit of the amide I component bands corresponding to surfactin and its diester are shown respectively in Fig. 3A,B. The methylation of surfactin produced an increase of the 1656-1657 cm l component band and a concomitant decrease of the 1642 c m - ~ component band, explaining the shift of the main band from 1648 cm t for surfactin to 1654 cm ~ for the diester. The methylation affected slightly the band shape in the amide II region but the main band was always located at 1537 cm ~ (Fig. 2 A - C , solid lines).

The infrared spectrum of surfactin in 2H20 shows two broad bands located at 1729 and 1712 cm l (Fig. 4A). The 1729 cm l component band corresponds to the C--O group of the cyclopeptide lactone ring. The 1712 cm L component band was assigned to the C O O H groups of aspartyl and glutamyl residues of surfactin and to the strongly hydrogen-bonded C=O group of the lactone ring. The shift to lower wavenumber from 1737 cm 1 (dry state, Fig. 2A) to 1729 cm ~ (hydrated state, Fig. 4A) and from 1718 c m - 1 (dry state, Fig. 2A) to 1712 cm 1 (hydrated state, Fig. 4A) may indicate the formation of hydrogen bonds involving water molecules. Methylation of the surfactin carboxylic groups produced a slight decrease in intensity of the 1712-1714 cm L component band and a concomitant increase in intensity of the high wavenumber component band at 1740 1742 cm ~, reflecting the conversion of - C O O H groups to - C O O C H 3 groups (Fig. 4B,C, solid lines). There was no wavenumber shift of the 1740-1742 cm ~ band of the 2H20-hydrated surfactin esters (Fig. 4B,C), as compared with the non-hydrated surfactin esters (Fig. 2B,C), reflecting the absence of hydrogen bonds between 2H20 and the C--O groups in the esters. The infrared spectrum of 2H20-hydrated surfactin contained at least four component bands located at 1679, 1659, 1641 and 1632 cm 1 in the amide I region as evidenced by its second derivative spectrum (Fig. 4A, dashed line). The result of curve fitting analysis in the amide I region containing these four component bands is shown in Fig. 5A. The component band at 1632 cm I was less resolved on the second derivative spectra of the mono- and di-ester of surfactin (Fig. 4B,C, dashed lines), suggesting that this component band is weaker in the surfactin derivatives. To better visualize these component bands and the different hydration steps, infrared difference spectra of surfactin and of its esters in 2H20 were measured after different incubation times. Positive bands on the infrared difference spectra showed the formation of new species with increasing time, while negative bands indicated the decrease of

G. Ferry; et al.

627

Spectrochimica Acta Part A 53 (1997) 62.] 635

A

B

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Fig. 3. Best-fined individual component bands of FTIR spectrum of surfactin (A) and its diester derivative (B). Their summations (dashed lines) were compared with the original spectrum measured in solid state (solid lines).

other species. The infrared difference spectra of surfactin, determined at increasing times, indicated an increase in intensity of the component bands at 1634, 1619 and 1450 cm ' and a concomitant decrease in intensity of the component bands at 1688, 1660 and 1554 cm ' (Fig. 6A). The negative 1688 and 1660 cm ~ component bands, as well as the positive 1634 and 1619 cm L component bands, correspond to the C=O group of the peptide backbone. The 1688 and 1660 cm ] component bands could be related to relatively free C=O groups, while the 1634 and 1619 cm -~ bands could correspond to hydrogenbonded C=O groups. Therefore the decrease of the 1688 and 1660 cm ~ component bands and the concomitant increase of the 1634 and 1619 cm l bands may reflect a shift of population from relatively free C=O groups to C ~ ) groups forming hydrogen bonds with 2H20 molecules. Alternatively, the hydration may induce slight conformational changes that affected the IR difference spectra of surfactin in the amide I region. Similar results were observed in the case of monoester (Fig. 6B) and diester surfactins (Fig. 6C) but the infrared spectral changes were less important. The negative 1541 1554 cm 1 band in the amide II region and the positive 1445 1452 cm " ' band in the amide II' region indicated the conver-

sion of N H groups of the peptide backbone to N2H groups and to the formation of HO2H, due to the exchange of the labile protons of surfactin with the deuterium of 2H20. The rate of H/2H exchanges of the N H groups with deuterium of the solvent was followed by measuring the integrated intensity of the 1400-1490 c m - ' broad component band. The exchange rate was much smaller in the case of the diester (Fig. 6C) than in the case of the monoester (Fig. 6B) or surfactin (Fig. 6A). For a better comparison, integrated intensity of the amide II' broad band was plotted as a function of time for each of these samples (Fig. 6D). The hydration-induced conformational change in surfactin and its derivatives was monitored as a function of time by determining the integrated intensity of the 1618-1648 cm ~ broad bands in the amide [ region (Fig. 6E). The hydration affected similarly the C O groups and the NH groups of surfactin since the changes in the amide I region (Fig. 6E, squares) paralleled the changes in the amide II' region (Fig. 6D, squares). However the C-O groups of the monoester and the diester (Fig. 6E, triangles and lozenges) hydrated faster than their NH groups (Fig. 6D, triangles and lozenges). These differences reflect the greater hydrophobicity of the monoester and the diester as compared with surfactin.

628

G. FerrO et al. , Spectrochimica Acre P a r t A 53 (1997) 623

3.3. Influence of the p H on the F T I R spectrum of surfactin, its monoester and diester derivatives The protonation state of the Asp and Glu residues was followed by F T I R spectroscopy as it had been shown that the C--O groups of C O O H absorb at 1712-1716 cm ~, while the C O O groups absorb at 1560-1574 cm J [24,44]. Fig. 7 A - D showed different steps of hydration of a surfactin film in the Tris buffer pZH 8.15. By comparing the IR spectrum of surfactin before hydration (Fig. 7A) with the spectrum measured after 5 min incubation in the buffer (Fig. 7C), the most important changes were a decrease of two bands at 1718 cm ~ and at 1537 cm ~ concomitant with the appearance of two bands at 1457

635

cm ~ and at 1559-1567 cm ~. The progressive disappearance of the 1537 cm 1 band and the related appearance of the 1457 cm ~ band corresponded to the H/2H exchanges of the N H groups previously observed in 2H20 (Fig. 6A). The decrease of the 1718 cm 1 band and the concomitant increase of the 1559 1567 c m - ~ band could be interpreted as deprotonation of the f l - C O O H of Asp and of the 7-COOH of Glu. Furthermore, the relatively more intense amide II' band after 20 min incubation in the buffer p2H 8.15 (Fig. 7D) as compared with the corresponding band in 2H20 (Fig. 6A) indicated more important H//2H exchanges of the N H groups. This is not surprising since surfactin is more soluble at alkaline p H and the H/2H exchange rate is pH-dependent [45].

3.4. FTIR spectra of protonated surJactin, its monoester and diester derivatives in different organic' solvents

Ol

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, 1400

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Fig. 4. F T I R spectra (solid lines) and second derivative spectra (dashed lines) of surfactin after a 20 rain incubation in 2H~O

(A) and of the monoester (B) and diester (C) derivatives after a 60 rain incubation in 2H20.

The use of organic solvents such as a hydrogen bond acceptor (DMSO) or a hydrogen bond donor (TFE) as well as the variation of temperature can provide more insight into the hydrogenbonded and non hydrogen-bonded species. In particular, the presence of various structures, involving distinct types of intramolecular hydrogen bonds, can be better evidenced by using organic solvents or temperature variation since the modification of concentration itself may not be sufficient for probing the presence of distinct conformers. The infrared spectrum of surfactin in T F E (Fig. 8A, solid line) and of its diester derivative in T F E (Fig. 8A, dashed line) were measured at temperature range from 20 to 40°C. No significant temperature-induced variations of the I R spectra either of surfactin or of its diester could be detected, indicating that the intramolecular hydrogen bonds were very strong. The IR spectrum of surfactin in T F E (Fig. 8A, solid line) shows a wide band centred at 1729 cm ~, while the I R spectrum of the diester surfactin indicates a narrower band centred at 1725 cm ~ (Fig. 8A, dashed line). Since T F E is a hydrogen bond donor solvent, it can form hydrogen bonds with the C - O of the lactone ring and with the C=O of the aspartic and glutamic acids, as well as with the

629

G. Ferry; et al. Spectrochimica Acta Part A 53 (1997) 623 635

g

0.1

=

A

0.1

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==

8 ,"

,

,"

(O'',

\

0 1700

1650

1600

Wavenumber (cm-1)

1700

1650

1600

Wavenumber (cm-1)

Fig. 5. Best-fined individual component bands of FTIR spectra of surfactin. Their summations (dashed lines) were compared with the original spectra (solid lines) measured after a 20 min incubation in -~H20 (A) or in DMSO (B).

C-~O groups of the peptide backbone of surfactin. The formation of hydrogen bonds between the C=O of the lactone ring of surfactin and T F E would explain the shift of the band from about 1737 cm-~ in dry state (Fig. 2A, solid line) to about 1729 cm ~ in T F E (Fig. 8A, solid line). Similarly, the formation of hydrogen bonds between the C=O of the lactone ring and TFE, as well as between the C=O of the aspartyl- and glutamyl-methylesters and TFE, would explain the shift of the surfactin diester bands from 1742 cm ~ in dry state (Fig. 2C) to 1725 c m 1 in T F E (Fig. 8A, dashed line). The main amide I band of surfactin shifted from 1648 cm ~ (dry state, Fig. 2A) to 1661 cm -~ (TFE, Fig. 8A, solid line). Smaller shift, from 1654 c m - ~ (dry state, Fig. 2C) to 1657 cm ~ (TFE, Fig. 8A, dashed line), was observed in the case of surfactin diester. The two component bands at 1737 and 1718 cm ~ observed in the IR spectrum of surfactin in dry state (Fig. 2A) coalesced into a single component band located at 1721 cm t on the IR spectrum of surfactin in DMSO (Fig. 8B, solid line), This suggests that there were more hydrogenbonded C--O groups of the lactone ring in DMSO than in dry state. This result is quite surprising because DMSO, a good hydrogen bond acceptor solvent, is a potent disrupter of hydrogen bonds involving the C-~) groups. Indeed DMSO breaks the hydrogen bonds of the C-O group of the

lactone ring in the diester surfactin since its IR spectrum in DMSO contains a single and narrower band located at 1737 cm 1 (Fig. 8B, dashed line), while the hydrogen-bonded C=O of the lactone ring of the diester surfactin in dry state give rise to a component band located at 1718 cm ] (Fig. 2C). The IR spectra of surfactin and of its diester in DMSO display a main amide I band located, respectively, at 1669 cm ~ (Fig. 8B, solid line) and 1665 cm 1 (Fig. 8B, dashed line). By comparing the IR spectra of both compounds in DMSO with the IR spectra of these compounds in dry state or after 2H20-hydration (Figs. 2 and 4), the main amide I band shifted to higher wavenumbers, indicating the presence of more free C=O groups in surfactin and its diester. The influence of the temperature on the IR spectrum of surfactin in DMSO was then studied, as the previous N M R study of the surfactin conformation, realized in DMSO, showed that the N H chemical shifts vary linearly with temperature and that the highest variation were observed for the NH of Leu2, Leu3 and Leu7 [20]. The IR spectra of DMSO and both lipopeptides, surfactin and its diester, were affected by heating from 20 to 60°C. The difference infrared spectrum of DMSO alone between 20 and 40°C shows a negative band at 1672 cm ~ (Fig. 9A). Beside this band, the difference infrared spectrum of surfactin in DMSO between 20 and 40°C (Fig. 9B) displays a negative

G. FerrO et al. : Spectrochimica Acta Part A 53 (1997) 623 635

630

band at 1648 cm ~ and positive bands appeared at 1654 and/or at 1696 cm ~. The decrease of the population of hydrogen-bonded C-O groups of the peptide backbone was signaled by the negative band at 1648 cm ~. The corresponding increase of less hydrogen-bonded C O species may explain the appearance of positive bands at higher wavenumbers (1654 or at 1696 cm ') (Fig. 9B). The difference infrared spectra of the surfactin diester in D M S O between 20 and 40°C (Fig. 9C) shows less variations than the difference infrared spectrum of surfactin realized under the same

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~

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;

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Fig. 7. FTIR spectra of the dry film of surfactin (A) and of the hydrated fihn just after addition of deuterated buffer (B), after 5 rain (C) or 20 rain incubation (D) in the buffer.

conditions. An intermediate state was obtained with the monoester (data not shown). Band-fitting analysis of the amide ! region of surfactin in D M S O at 20°C was then realized by using the component band positions determined with the second derivative spectrum. By comparing the curve fitting analysis (Fig. 5B) with the influence of temperature (Fig. 9C), it appeared that the 1672 cm ~ component band corresponding to non-hydrogen bond C=O groups was not affected

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Fig. 6. FTIR difference spectra of surfactin in 2H20 (A) after 10 min (...), 15 rain ( - - ) or 20 rain ( ) of incubation. (B) corresponds to the IR difference spectra of the monoester and (C) to the diester after 20 min ( - - ) , 4 0 r a i n ( • ) o r 6 0 m i n ( • • ) of incubation. (D) gives the increase of the integrated positive peaks in the amide lr region (1400 1490 cm ~) and (E) gives the increase of the integrated positive peaks in the amid• I region (1618 1648 cm 1).

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Fig. 8. FTIR spectra of surfactin (solid lines) and its diester derivative (dashed lines) in TFE at 20°C (A) or in D M S O at 20°C (B).

G. Ferr~ eta/.

Spectrochimica Acta Part ,4 5,7 (1997) 623 635

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Fig. 9. influence of temperature on the surfactin conformations in DMSO. IR difference spectra between 20 and 40°C of DMSO alone (A), of surfactin (B) and its diester derivative (C) in DMSO.

by the temperature increase. The 1648 1649 cm 1 component band of surfactin, corresponding to C-~) involved in hydrogen bonds with N H groups, decreased with temperature increase. The very small 1696 cm-~ component band did not contribute significantly to the curve fitting and was omitted in Fig. 5B.

4. Discussion 4.1. Interpretation o f infrared spectra o f surfactin, its monoester and diester deriv'atives in the 1700 1750 cm - 1region

The 1700 1750 c m ~ region is characteristic of the C=O stretching mode of esters or carboxylic acids of aspartyl or glutamyl residues [24]. We propose that the 1729 1737 cm t component band corresponds to relatively free (i.e. non-hydrogen bonded) C=O group of the lactone ring in surfactin while the 1712 1725 cm l component band is related to the C=O group of the lactone ring hydrogen-bonded either with the solvent molecules or with other polar group of the lipopeptide. This interpretation is consistent with the shift of the 1737 cm ~ J band for surfactin in dry state to 1729 cm ' for surfactin in hydrated state or in TFE, where the solvent molecules can form hydrogen bonds with the C ~ ) groups of the lactone ring. Such a shift was also observed when surfactin was dissolved in DMSO. In this case, the C=O group of the lactone ring absorbs at 1721

631

cm ~, i.e. much lower wavenumber value than the expected value for an acceptor hydrogen bond solvent such as DMSO. These unexpected results can be explained either by DMSO-induced conformation changes, promoting the formation of hydrogen-bonds between the C-O groups of the lactone ring and other hydrogen donors in the lipopeptide (inter or intramolecular interactions), or by the formation of hydrogen bond between the C=O groups of the lactone ring and water molecules, present as traces in DMSO, or by both of these interpretations. Such interactions decreased or disappeared for diester surfactin in DMSO since its IR spectrum showed one single component band at 1737 c m - ' . The C=O groups of the aspartyl and glutamyl residues in protonated surfactin gives rise to a band around 1712 1725 cm ~, which overlaps the band related to the C--O group of the lactone ring. Variation of pH unambiguously identified the COOH groups of the aspartyl and glutamyl residues since the C O 0 groups absorb at 15591567 cm 1 while the COOH groups absorb at around 1700-1730 cm ~. The esterification of these two carboxylic groups produced a new band at 1737-1742 cm ~, associated with the COOCH3 groups, overlapping the band corresponding to the C - O group of the lactone ring. These COOCH 3 groups can form hydrogen bonds with the solvent molecules as evidenced by the shift of the band from 1742 cm ~ (dry state) to 1725 c m ] (TFE) in the case of the surfactin diester.

4.2. Interpretation o f infrared spectra o f surJ'actin in the 1600-1700 cm - I region

There are two types of carbonyl groups which could absorb in the 1600-1700 c m - ] region. The first type corresponds to the C-O group of the fatty acid adjacent to Glul (Fig. 1) and the second type is related to the six C=O groups of the peptide backbone. Both types of carbonyl groups were treated as amide I vibrational modes as they were in the case of C H 3 - C O ( A I a ) 4 - N H - C H 3 [47]. The seventh C=O group of the Leu7 formed the lactone ring and was already discussed above.

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Spectrochimica Acta Part A 53 (1997) 623 635

The infrared spectrum of surfactin in dry state contained at least three component bands located at 1681 cm -~ (medium), 1657 cm (strong) and 1643 cm ~ (very strong) in the amide I region (Fig. 3A). This infrared spectrum of surfactin presented some similarities observed on other spectra of fl-turn containing peptides, such as G l y - L - P r o - G l y - G l y with three component bands located at 1697 cm -~, 1654 c m - l and 1647 c m - J [47] or as iturin, a cylo L,Dlipopeptide, with four component bands at 1692 cm I (weak), 1665 cm ~ (weak), 1654 cm (strong) and 1639 c m - ~ (very strong) [39]. It was earlier noted that while c~-helix and fl-sheet component of proteins may be expected to have relatively characteristic and constant amide I wavenumbers, the same is not likely true of a fl-turn component. Indeed large variation of wavenumber shift were predicted (from 16901675 cm l) for a given type I fl-turn with respectively different dihedral angles (from - 6 0 to - 75 °) [23,46]. To ascertain the presence of fl-turn in surfactin, two relatively specific characteristics of fl-turn were searched. Recently it was proposed that the intramolecular hydrogen bonded C-~O group involved in fl-turn of cyclic hexapeptides gives rise to characteristic amide I band in the range of 1638-1646 cm ~, with the exact position depending on the solvent and the side chains [41]. Such a component band was found on the IR spectrum of surfactin at 1643 cm l (dry state), 1641 cm ~ (hydrated state) or 1648-1649 cm ~ (DMSO). The variation of temperature from 20 to 40°C of surfactin in D M S O induced small changes in intensity of this 1648 1649 cm ~ component band, that was consistent with the assignment of hydrogen bonded C--O groups. The second characteristic for probing a fl-turn was evidenced by using D M S O as a good disruptor of hydrogen bonds involving C--O groups which kept intact the intramolecular fl-turn hydrogen bond [38-41]. The non hydrogen bonded C=O groups should appear at 1660-1670 cm l range [38,41,48]. The infrared spectrum of surfactin in D M S O showed three component bands located at 1672, 1659 and 1649 cm ~ Comparisons of this in-

frared spectrum with the infrared spectra of surfactin in dry state or in 2H20 indicated that D M S O promoted an increase of the 1672 1681 cm l component band at the expense of the 1657-1659 and 1641 1649 cm -~ component bands. We propose that the part corresponding to these component bands which were affected by the addition of D M S O may reflect the external hydrogen bonded C-~2) groups or other types of C=O groups, while the remaining component band located at 1641-1649 cm -1 on the infrared spectrum of surfactin in D M S O may reflect the intact hydrogen bonded C--O group involved in fl-turn. The presence of such a hydrogen bond between the N H of Leu2 and the C-~3 of Asp5 was proposed on the basis of N M R data on surfactin in D M S O [20], more precisely the N H chemical shift ( 6 N H ) was measured as function of temperature (T) and the temperature coefficients (A6NH/AT) of N H protons for Leu2, Leu3 and Leu7 were the highest. This N M R study did not excluded other types of structures for surfactin and, in fact, two different conformers S1 and $2 were proposed [20]. The $2 structural model of surfactin contained two 7-turns (one including the N H of Leu7 and the C=O of Asp5 and the other the N H of Val4 and the C O of Leu2) and one hydrogen bond (between the N H of Leu6 and the C=O of the fl-hydroxy fatty acid). The infrared spectrum of cyclo (D-Phe L - P r o - G I y - D - A l a - L - P r o ) , a 7,turn peptide model, contained four component bands located at 1689, 1666 (the most intense), 1638 and 1621 cm ~ [49]. The 1621-1625 cm -~ band may signal the presence of ~,-turn hydrogen-bonded C=O group [40,42,49]. Even if this component band was not very well evidenced on our infrared spectra, the presence of y-turns in surfactin cannot be excluded from our F T I R spectra. Based on this interpretation, one may conclude that ?,-turns are not predominant since the 1621-1625 c m - ~ component band was relatively weak or was not detected. However a cautious approach is necessary in the case of ),-turns since the characteristic amide I bands for 7-turns is not well established as for fiturns. Furthermore transition dipole coupling effects may split and shift some component bands, complicating the spectral analysis [23].

G. Ferrb et al. Spectrochimica Acta Part ,4 53 (1997) 623 635 4.3. Influence o f the methylation o f the carboxyl residues on the spectra o f monoester and diester sutJi~ctins in the 1600-1700 cm ~ region

The N M R study combined with molecular modeling of surfactin suggested an important role for the two polar Glu and Asp side chain residues (located on the opposite of the aliphatic chain of the fl-hydroxy fatty acid residue) in the surfactin conformation [20]. The methylation of aspartyl and glutamyl residues in surfactin modifies the IR spectrum in the 1700-1750 cm-~ region as it was discussed above. Such modifications may indirectly induce conformational changes of the peptide backbone of the lipopeptides, which could be detected in the 1600-1700 cm -~ region. The methylation of only the glutamyl residue of surfactin produced a shift of the amide I band from 1648 (surfactin in dry state) to 1650 cm ~ (surfactin monoester in dry state) or from 1644 (surfactin in 2I-I20) to 1646 cm ~ (surfactin monoester in 2H20 ). The dimethylation of surfactin leaded to a more pronounced shift of the amide I band from 1648 (surfactin in dry state) to 1654 cm ~ (surfactin diester in dry state) or from 1644 (surfactin in 2H20) to 1648 cm ~ (surfactin diester in 2H20). These shifts of the amide I band toward higher wavenumber produced by the methylation of surfactin is consistent with a change of populations from hydrogen-bonded species to less hydrogen-bonded or free species. However when T F E or DMSO were used instead of 2H20, the amide I band shifted to lower wavenumber, i.e. from 1661 (surfactin in TFE) to 1657 cm (surfactin diester in TFE) or from 1669 (surfactin in DMSO) to 1665 cm i (surfactin diester in DMSO). This indicates that organic solvents induced less hydrogen-bonded species involving C=O groups of peptide backbone of surfactin than those of diester. The H/2H exchange rate of the N H was reduced when surfactin was methylated or dimethylated. This methylation-induced decrease of the H/2H exchange rate is consistent with the greatest hydrophobicity of both ester derivatives of surfactin.

633

5. Conclusions Our IR results corroborate the previous use of the amide I component bands as a mean to characterize /J-turns in cyclic peptides [40 42]. In particular, the 1641 1649 cm 1 component band, assigned to hydrogen-bonded C-~) groups involved in fl-turn structures, may constitute a characteristic amide I component band. Furthermore the use of organic solvents could enhance the diagnostic value by indicating the exposed hydrogen bonds involving C=O residues. In this respect, DMSO, which can break the exposed hydrogen bonds involving C=O and can preserve fl-turn hydrogen bonds, potentiates the identification of fl-turns. Such a remaining component band at 1641-1649 c m - 1 was observed in the IR spectrum of surfactin in DMSO, pointing out the presence of fl-turn in the lipopeptide. Furthermore the 1672 cm ~ component band on the IR spectrum of surfactin in DMSO (corresponding to relatively free C=O groups) absorbed at the same wavenumber range (1673 cm ~) in cyclic hexapeptides containing fl-turns [41]. This result is consistent with the previous N M R study of surfactin in DMSO, suggesting the presence of a fl-turn in S1 conformer [20]. However this study also proposes another conformer ($2) containing ;,-turns. The presence of ).-turns in surfactin cannot be ascertained from our IR results, since the characteristic amide I bands of 7-turns are not well documented. Although the fl-turns were always detected in surfactin as well as in its monoand di-ester derivatives, the methylation of surfactin altered slightly the conformation of the lipopeptide or/'and its relative population of conformers. These small structural alterations observed in the amide I region of both surfactin esters, as well as in their 1700-1750 cm ~ region, could explain the modifications of the biological and surfactant properties induced by the monoand di-methylation of surfactin [12,21]. The hydrophobicity, the peptide backbone conformation and the ability of the C--O group of the lactone to form hydrogen bonds with solvent molecules or with polar groups of peptide moiety are the three important structural parameters which are different in surfactin and in ester derivatives. It was

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s u g g e s t e d t h a t t h e i n c r e a s e o f h y d r o p h o b i c i t y ind u c e d by t h e m e t h y l a t i o n o f s u r f a c t i n c o u l d explain the s t r o n g i n c r e a s e o f h a e m o l y t i c a c t i v i t y [21]. W e p r o p o s e that, in a d d i t i o n to t h e c h a n g e of hydrophobicity, change of peptide backbone c o n f o r m a t i o n m a y a l t e r the h a e m o l y t i c activity. Moreover, the inhibition of the cAMP phosphodiesterase b y a s u r f a c t i n w i t h a n o p e n e d l a c t o n e l i n k a g e was h a l f o r less t h a n t h a t o f t h e p a r e n t s [12]. It is t e m p t i n g to relate the d i f f e r e n c e in the i n h i b i t i o n o f c A M P p h o s p h o d i e s t e r a s e [12] to t h e d i f f e r e n c e in the ability o f f o r m a t i o n o f h y d r o g e n b o n d s ( o b s e r v e d by I R s p e c t r o s c o p y ) i n v o l v i n g C = O g r o u p s o f l a c t o n e m o i e t y in s u r f a c t i n a n d in its d e r i v a t i v e . F u r t h e r w o r k is n e e d e d to precise t h e n a t u r e a n d the b a l a n c e o f these s t r u c t u r a l e l e m e n t s for a b e t t e r u n d e r s t a n d i n g o f t h e differe n t activities o f s u r f a c t i n a n d its d e r i v a t i v e .

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