Langmuir 2009, 25, 4211-4218
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Aggregation of the Naturally Occurring Lipopeptide, Surfactin, at Interfaces and in Solution: An Unusual Type of Surfactant?† Hsin-Hui Shen,‡ Robert K. Thomas,*,‡ Chien-Yen Chen,§ Richard C. Darton,§ Simon C. Baker,| and Jeffrey Penfold⊥ Physical and Theoretical Chemistry Laboratory, South Parks Road, UniVersity of Oxford, Oxford OX1 3QZ, U.K., Department of Engineering Science, Parks Road, UniVersity of Oxford, Oxford OX1 3PJ, U.K., School of Life Sciences, Oxford Brookes UniVersity, Gypsy Lane, Oxford, OX3 0BP, U.K., and ISIS, STFC, Rutherford-Appleton Laboratory, Chilton, Didcot, Oxon OX11 0QX, U.K. ReceiVed September 5, 2008. ReVised Manuscript ReceiVed December 3, 2008 Neutron reflectometry has been used to study the structure of the biosurfactant, surfactin, at the air/water and at the hydrophobic solid/water interfaces. Three different deuterated surfactins were produced from the Bacillus subtilis strain: one perdeuterated, one with the four leucines perdeuterated, and one with everything except the four leucines perdeuterated. The neutron reflectivity profiles of these three samples in null reflecting water and in D2O with a seventh profile of the protonated surfactin in D2O were measured at pH 7.5. This combination of different isotopic compositions made it possible to deduce the distribution of each type of labeled fragment in the surfactin. Surfactin is found to adopt a ball-like structure with a thickness of 14 ( Å and an area per molecule of 147 ( 5 Å2. This makes it more like a hydrophobic nanoparticle, whose solubility in water is maintained only by its charge, than a conventional surfactant. This is probably what makes it surface-active at such low concentrations and what contributes to its forming very compact surface layers that are more dense than observed for most conventional amphiphiles. The reflectivity data were fitted by a model in which the structure of surfactin was divided into three fragments: the four leucines taken as a group, the hydrocarbon chain, and a hydrophilic group containing the two negative charges. An analysis of the reflectivity gave the following separations between fragments, where zero corresponds to the Gibbs plane for zero water adsorption: chain-water 7 Å, hydrophile-water 1 Å, and leucines-water 6.5 Å, all (1 Å. The overall structure of the layer appears to be identical at a hydrophobic octadecyltrichlorosilane-coated silicon surface where the thickness of the surfactin layer is 15 ( 1 Å and the area per molecule is 145 ( 5 Å2. Finally, the structure of surfactin micelles has been examined by means of small-angle neutron scattering. The aggregation number was found to be unusually small at 20 ( 5. The structure of the micelle is of the core-shell type with the hydrocarbon chain and the four hydrophobic leucines forming the core of the micelle.
Introduction Understanding the interfacial properties of naturally occurring biosurfactants not only helps in understanding their biological role but also is of considerable importance for applications in medicine (e.g., antibacterials or antibiotics) and in the development of sustainable products (e.g., home and personal care products). Although there is no difficulty in identifying suitable species for such investigations because of the wide range of materials and material types that have been identified,1,2 such studies are severely limited because of the lack of experimental techniques available for simultaneously determining composition and structure at wet interfaces. At present, neutron reflectometry is a technique that is very effective in providing simultaneous information about both the composition and structure of a layer but is only at its most effective if deuterium-labeled compounds are available, which is not usually the case for naturally occurrring biomolecules. Many biosurfactants are assumed to have the typical characteristics of simple amphiphilic molecules because, like conventional amphiphiles, they both aggregate in solution and † Part of the Neutron Reflectivity special issue. * Corresponding author. E-mail:
[email protected]. ‡ Physical and Theoretical Chemistry Laboratory, University of Oxford. § Department of Engineering Science, University of Oxford. | Oxford Brookes University. ⊥ ISIS.
(1) Carrillo, P. G.; Mardaraz, C.; PittaAlvarez, S. I.; Giulietti, A. M. World J. Microbiol. Biotechnol. 1996, 12, 82. (2) Benincasa, M.; Abalos, A.; Oliveira, I.; Manresa, A. Antonie Van Leeuwenhoek 2004, 85, 1.
are surface-active at low concentrations. However, because their structures are more complex it is not always easy to identify clearly the hydrophobic and hydrophilic regions of the molecule, and experimental information about either aggregation or adsorption is scarce, although it is of considerable importance for understanding their function and potential applications.3,4 Surfactin is just such an example. The molecule consists of a long-chain R-hydroxy fatty acid whose two functional groups close a short peptide chain of seven amino acid residues (Figure 1). The residues are four leucines (two D- and two L-), one valine, and one each of glutamic and aspartic acid. Bonmatin et al.5 have used 1H NMR to show that in DMSO the peptide ring of surfactin adopts two possible structures, designated S1 and S2. S2 is a saddlelike structure with the two charged acid residues forming a bidentate group that is a potential binding site for divalent cations. Additional computer simulations at a hydrophilic/ hydrophobic interface6 provided possible conformations based on Bonmatin’s simulation but including the side chain. These and NMR results have found that the S2 conformation with a folded fatty acid chain gives a more consistent interfacial area per molecule compared with those obtained from pressure-area (Π-A) isotherms. Surfactin is also very surface-active with a (3) Rodrigues, L.; Banat, I. M.; Teixeira, J.; Oliveira, R. J. Antimicrob. Chemother. 2006, 57, 609. (4) Shete, A. M.; Wadhawa, G.; Banat, I. M.; Chopade, B. A. J. Sci. Ind. Res. 2006, 65, 91. (5) Bonmatin, J. M.; Genest, M.; Labbe, H.; Ptak, M. Biopolymers 1994, 34, 975. (6) Gallet, X.; Deleu, M.; Razafindralambo, H.; Jacques, P.; Thonart, P.; Paquot, M.; Brasseur, R. Langmuir 1999, 15, 2409.
10.1021/la802913x CCC: $40.75 2009 American Chemical Society Published on Web 01/08/2009
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Figure 1. Chemical structure of the heptapeptide ring of surfactin.5
very low critical micelle concentration (cmc). It is a strong foaming agent and a powerful emulsifier7,8 and exhibits a strong membrane destabilizing action. However, although studies of adsorbed surfactin have been made by several techniques such as IR spectroscopy,9,10 NMR,5,11 circular dichroism,12,13 and computer simulation,6,14 none of these gives direct information about its interfacial structure or even interfacial density. Neutron reflectometry is capable of giving structural and compositional information about an interface if differences in the neutron refractive index can be created between different parts of either or both amphiphile and solvent. The difference in refractive index (contrast) between hydrogen- and deuterium-containing materials is very large, and the technique can be brought to bear on the issue of the interfacial structure of surfactin layers if deuterated samples can be made available. The production of surfactin from bacteria and the design of four different neutron contrast experiments made use of the known surfactin biosynthesis pathway.15 The biosynthesis of perdeuterated and partially deuterated samples is generally nontrivial, but we have successfully synthesized three different deuterated samples of surfactin by changing the culture conditions between H2O and D2O, d- and h-glucose, or d- and h-leucine (d denotes perdeuteration). These samples have enabled us to determine the structure and surface density of surfactin monolayers at two surfacessair/water and hydrophobic solid/watersand to identify the structure of a surfactin micelle. Figure 2 shows the structure and deuterium-labeling scheme in these samples, including a possible orientation of the hydrocarbon chain whose conformation was not determined in the original NMR study of Bonmatin et al.5
Experimental Methods Surfactin Production. Surfactin is unsuitable for synthesis by the Fmoc solid-phase peptide synthesis method, often used for isotopically labeled samples, in part because of the presence of a long fatty acid chain in the molecule. Thus, Pagadoy et al.16 have (7) Deleu, M.; Razafindralambo, H.; Popineau, Y.; Jacques, P.; Thonart, P.; Paquot, M. Colloids Surf., A 1999, 152, 3. (8) Razafindralambo, H.; Paquot, M.; Baniel, A.; Popineau, Y.; Hbid, C.; Jacques, P.; Thonart, P. Journal of the American Oil Chemists Society 1996, 73, 149. (9) Vass, E.; Besson, F.; Majer, Z.; Volpon, L.; Hollosi, M. Biochem. Biophys. Res. Commun. 2001, 282, 361. (10) Ferre, G.; Besson, F.; Buchet, R. Spectrochim. Acta, Part A 1997, 53, 623. (11) Heerklotz, H.; Seelig, J. Biophys. J. 2001, 81, 1547. (12) Osman, M.; Ishigami, Y.; Ishikawa, K.; Ishizuka, Y.; Holmsen, H. Biotechnol. Lett. 1994, 16, 913. (13) Osman, M.; Hoiland, H.; Holmsen, H. Colloids Surf., B 1998, 11, 167. (14) Nicolas, J. P. Biophys. J. 2003, 85, 1377. (15) Steller, S.; Sokoll, A.; Wilde, C.; Bernhard, F.; Franke, P.; Vater, J. Biochemistry 2004, 43, 11331.
Figure 2. (a) Possible structural arrangement of the surfactin skeleton: C in the ring (black), C in the chain (gray), O (red), N (green), and O(magenta). The hydrogen atoms are not shown. The ring has the structure deduced by Bonmatin et al.,5 and the chain has been arbitrarily chosen to point away from the ring. (b, c) Schematic of the appearance of two partially deuterated surfactin molecules with respect to the neutron beam. The structure is the same as in structure a, with all of the atoms in the CH2 regions colored blue and all of those in the CD2 regions colored red. (b) Structure designated S-H-H-d-Leu and fully protonated except for the four leucine residues. (c) Structure designated S-D-D-h-Leu and fully deuterated apart from the four leucine residues.
tried to synthesize surfactin by combining the Fmoc method with more traditional chemical synthesis, but the yields have so far been low and the synthesis is very time-consuming. Here, we chose to
Aggregation of Surfactin
Langmuir, Vol. 25, No. 7, 2009 4213 Table 1. Summary of the Four Isotopic Surfactin Compositions from ESI-MS
conditions
S-H-H
S-D-D
S-H-H-d-Leu
S-D-D-h-Leu
ESI-MS surfactin
1088 1022 1036
1088 1102 1120
1044 1058 1072
1043 1058 1075
no. of deuterium atoms
0 0 0
80(80) 80(82) 84(84)
36(40) 36(40) 36(40)
35(40) 36(42) 39(44)
deuterium no. (%)
0
98
90
88
Table 2. Calculated Scattering Length Densities for the Four Isotopic Surfactin Compositions surfactin contrasts -1
M/g mol solvent b/10-5 Å-1 V/Å3 F/10-6 Å-2
S-H-H 1036.38 NRW 15.32 1516 1.01
D2O 24.69 1.63
S-D-D 1120.38 NRW 97.84 1516 6.45
avoid the complexity and cost of the synthesis and produced surfactin directly from bacteria. The strain BBK006 was used for surfactin production.17,18 A single colony of bacteria grown on an LB agar plate was added to M9 medium (with 0.2% glucose) as a seed culture and made up to 5 mL in a 50 mL flask. This was incubated for 24 h on an orbital shaker (200 rpm) at 30 °C. A 10% volume of seed culture was used for further inoculation and growth. The seed culture for the deuterated syntheses was produced using an M9 medium containing 30% D2O and h-glucose as the carbon source. After incubation for 48 h, the partially deuterated bacteria were harvested using centrifugation at 5000 rpm for 15 min. A pellet of this seed culture was added to 50 mL of M9 media made from 100% D2O containing 0.2% deuterated glucose. Growth under these circumstances was rapid and led to surfactin with high levels of deuteration (approximately equal to the original D2O purity of 98%). The LB agar was prepared in six steps: (i) addition of 10 g of bacto-tryptone, 5 g of yeast extract, and 10 g of NaCl to 800 mL of H2O, (ii) adjustment of pH to 7.5 with NaOH, (iii) addition of 15 g of agar, (iv) melting of agar into the solution in a microwave oven, (v) adjustment of the volume to 1 L with H2O, and (vi) sterilization by autoclaving. The composition of the M9 medium is 0.2% glucose in 1 g/L NH4Cl, 3 g/L KH2PO4, 6 g/L Na2HPO4, 5 g/L NaCl, 1 mM MgSO4, and 0.1 mM CaCl2. The pH was adjusted to 7.0 with 0.5 M NaOH and sterilized at 121 °C for 20 min. All mineral salts were from Sigma-Aldrich and bactotryptone, and yeast extract and agar were from DIFCO Microbiology. The separation and purification of surfactin has been described previously.5,17 We also explored the ability of the bacteria to save energy by utilizing amino acids provided as feedstock in the culture medium. Thus, we added protonated or deuterated DL-leucine to the culture medium to produce samples with deuteration in different parts of the molecule. This was found to be completely effective. We therefore finished with four forms of surfactin: fully protonated, (S-H-H), fully deuterated apart from exchangeable protons (S-D-D), fully deuterated apart from the four leucines (S-D-D-h-Leu), and fully protonated apart from the four perdeuterated leucines (S-H-H-dLeu). The deuterated leucine was synthesized from isocaproic acid, which was perdeuterated by direct exchange with D2O,19 brominated in the R-position, followed by amine substitution using standard reactions. The level of deuteration and the localization of the labels within the molecule were characterized by mass spectroscopy. The molecular weight was determined using data collected from an LCT mass (16) Pagadoy, M.; Peypoux, F.; Wallach, J. Int. J. Pept. Res. Ther. 2005, 11, 195. (17) Chen, C. Y.; Baker, S. C.; Darton, R. C. J. Chem. Technol. Biotechnol. 2006, 81, 1923. (18) Chen, C. Y.; Baker, S. C.; Darton, R. C. J. Chem. Technol. Biotechnol. 2006, 81, 1915. (19) Zimmermann, H. Liq. Cryst. 1989, 4, 591.
S-H-H-d-Leu
D2O 108.03 7.13
1076.38 NRW 52.82 1516 3.48
D2O 62.19 4.10
S-D-D-h-Leu 1080.38 NRW 61.15 1516 4.03
D2O 70.54 4.65
spectrophotometer (Micromass). After the final product was freeze dried, it was dissolved in 50:50 MeOH/CH2Cl2 at 1 mg/mL and then diluted 50- to 100-fold in MeOH. The solution was subjected to electrospray ionization mass spectrometry (ESI-MS) in negative mode with medium-high (5,000) resolution. The ESI-MS results showed three main distribution peaks at 1008, 1022, and 1036 g/mol. The mass spectrometry of commercial surfactin was also compared as a reference. Surfactin purchased from Sigma with 99% purity showed exactly the same distribution peaks in the ESI-MS spectrometry measurement. To confirm the existence of a single product, the molecular weight of the fatty acid was examined following hydrolysis. The process of acid hydrolysis destroys the peptide moiety of surfactin. Following extraction of the hydrolysis products with CHCl3, the nonpolar part of the β-hydroxy fatty acid chain was collected for electron spray mass spectroscopy. The GCMS results showed three main peaks at 230, 244, and 258 g/mol corresponding to the β-hydroxy fatty acid chains of surfactin A, B, and C with 11, 12, and 13 carbons in the fatty acid chain, respectively. The level of deuteration in perdeuterated surfactin was that of the starting D2O (i.e., 98%), and in the partially labeled leucine species it was 90%, which corresponded to the level of deuteration in the starting leucine. Figure 2 schematically represents the deuterium content of each composition. The deuterium percentages are listed in Table 1, and the calculated scattering length densities for the four surfactin contrasts are presented in Table 2 and are based on the results in Table 1 and values of the volumes of the individual fragments taken from the literature.20,21 Although there are three surfactin analogues in the mixture with differing β-hydroxy fatty acid chains, we found that the variation in the scattering length density between the three species was slight. Because the C13 acid is dominant in the mixture, the scattering length densities in Table 2 are based on this single variant. Generally the calculated scattering length densities are in the order of S-D-D . S-D-D-h-Leu > S-H-H-d-Leu . S-H-H. For the air/liquid interface studies, two solution contrasts were used: NRW(null reflecting water) and D2O. Therefore, there are eight different combinations of scattering length density listed in Table 2. This Table takes into account hydrogen/deuterium exchange on the amino acids. Of the eight possible contrasts, the scattering length densities of S-H-H and NRW are fairly similar, and the S-H-H/NRW contrast was omitted. Seven contrasts should be sufficient to characterize the surfactin structure at the air/water interface by means of neutron reflectometry. Other Experimental Methods. The neutron reflectivity measurements were made on the SURF reflectometer at the ISIS neutron source UK.22 The measurements were made using a single detector at a fixed angle of θ ) 1.5° using neutron wavelengths in the range (20) Zamyatnin, A. A. Annu. ReV. Biophys. Bioeng. 1984, 13, 145. (21) Jacrot, B. Rep. Prog. Phys. 1976, 39, 911. (22) Penfold, J. Physica B 1991, 173, 1.
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of 0.5-6.8 Å-1 to provide a momentum transfer range of 0.048-0.5 Å-1, with the momentum transfer Q being defined as Q ) 4π sin θ/λ. Reflectivity profiles were fitted using a Java program based on the kinematic approximation.23,24 The reflectivity R is given as a function of Q by
R(κ) )
( )[∑ 16π2 Q2
bi2hii +
∑ ∑ 2bibjhij]
(1)
where hii and hij are the partial structure factors of the different fragments. The hii’s are the squared Fourier transforms of the individual fragment distributions normal to the surface, and the hij’s are products of the Fourier transforms of fragments i and j. The Fourier transforms of the fragments were determined numerically, and the distributions are described below. The final reflectivity was obtained from eq 1 by the application of a correction devised by Cowley25 that essentially allows a conversion from the approximate kinematic reflectivity of eq 1 to the exact value. This method of analysis has been widely used elsewhere and is fully described by Lu et al.26 The small-angle scattering experiments were conducted on the D11 reflectometer at the Institut Laue Langevin, Grenoble, France.27 The data were corrected for transmission and solvent background and converted to absolute intensity using standard procedures. The analysis used the core-shell model, similar to that widely used elsewhere.28 The form factor is expressed as
F(Q) ) V1(F1 - F2)F0(QR1) + V2(F2 - F3)F0(QR2) (2) where V1 and V2, F1 and F2, and F1 and F2 are the volumes, scattering length densities, and form factors of the inner volume (core) and outer shell, respectively. Q is the momentum transfer. The solid/liquid interface experiments used silicon single crystals of dimensions 125 × 50 × 25 mm3 that were polished on the (111) face. The experimental solution was held in a Teflon container with a volume of approximately 13 mL, which was clamped against the silicon block between temperature-controlled aluminum and magnetic stirrer plates. Sample changes were made through inlet and outlet ports located on opposite sides of the container, which could be connected to plastic tubes for injection by syringe. Stirring of the solution was achieved by a magnetic flea placed in a compartment in the wall of the trough and a small magnetic stirrer plate clamped behind the sample housing. The main advantage of this design is that stirring ensures the rapid attainment of equilibrium. After the silicon block was cleaned by UV/ozone treatment, hydrophobic surfaces for neutron reflection experiments were obtained by immersing the silicon blocks in a Teflon reaction vessel containing 2 × 10-3 M perdeuterated octadecyl trichlorosilane (dOTS) in n-hexadecane (+99% Sigma, without further purification) at 20 °C overnight. Upon removal of the blocks, the excess d-OTS was removed by wiping the surface with Kodak lens paper soaked in dry dichloromethane, followed by wiping with ethanol to remove the dichloromethane and finally rinsing with UHQ water. Following several sequential rinses with dichloromethane, ethanol, and UHQ water until the residual white patches on the surface, caused by polymerized OTS in the bulk solution, were removed, the hydrophobic layer was robust enough to withstand rubbing without introducing scratches or other defects detrimental to neutron reflection experiments. d-OTS (isotopic purity 92%) was synthesized from perdeuterated octadecyl bromide as described elsewhere.29 (23) Crowley, T. L.; Lee, E. M.; Simister, E. A.; Thomas, R. K.; Penfold, J.; Rennie, A. R. Colloids Surf. 1991, 52, 85. (24) Crowley, T. L.; Lee, E. M.; Simister, E. A.; Thomas, R. K. Physica B 1991, 173, 143. (25) Crowley, T. L. Physica A 1993, 195, 354. (26) Lu, J. R.; Thomas, R. K.; Penfold, J. AdV. Colloid Interface Sci. 2000, 84, 143. (27) Institut Laue-LangeVin: Neutron facilities at the high flux beam reactor, 1994. (28) Penfold, J.; Staples, E; Ugazio, I., S; Tucker;Soubiran, J., L.; Hubbard; Noro, M.; O’Malley, B.; Ferrante, G., A.; Ford; Buron, H. J. Phys. Chem. B 2005, 109, 18107.
The surface activity of surfactin was characterized by surface tension measurements at 298 K using a Kruss K10 tensiometer with a Pt-Ir ring. The water was ultrapure (ElgaStat), the ring was cleaned in a flame before each measurement, and all glassware and Teflon troughs were washed in neutral Decon and thoroughly rinsed before use. Measurements were made under several conditions. For the purposes of this article, we quote only results at pH 7.5. The cmc was determined from the abrupt change in slope in the plot of surface tension against the natural log of the concentration.
Results and Discussion Neutron Reflection. The sensitivity of neutron reflectometry to the structure of an adsorbed layer depends on its contrast with the surrounding medium and contrast differences within the molecule.30 Contrast is mainly a function of the hydrogen/ deuterium ratio and density. In general, the reflection of neutrons from a thin film gives rise to interference fringes whose separation is determined by the film thickness and whose amplitude is determined by the differences in scattering length density in the layer. The neutron scattering length density profile, F(z), across an adsorbed layer is defined as
F(z) )
∑ ni(z) bi
(3)
i
where ni is the number density of nucleus i, bi is its empirically known scattering length, and z is the direction along the normal to the surface. The scattering length b is very different for H and D, and this leads to two useful limiting contrasts. In the first, the H/D ratio in the water is adjusted so that its scattering length density is identical to that of air. There is then no reflected signal at all from the water (NRW). Apart from a weak background, the signal is specifically from the adsorbed layer and will be large if the layer is partially deuterated and moderately dense. Figure 3a shows the reflectivity profiles for the three deuterated surfactins in NRW at a pH of 7.5 and at the cmc of surfactin at that pH (6 × 10-6 M). As would be expected, the largest signal is from the perdeuterated sample. Taking the known scattering lengths of the three isotopically labeled surfactins and fitting the layer as a Gaussian distribution normal to the surface gives the result that in all three cases the area per molecule at the surface is 147 ( 10 Å2 and the overall thickness of the layer is 14 ( 2 Å, defined as σ in
f(z) )
( ) ( ) 2 4z2 exp - 2 σ σA√π
(4)
where the prefactor of the Gaussian is a normalizing factor. In this equation, A is the area per molecule, z is the distance along the surface normal, and σ is the width of the distribution at 1/e of the maximum. The calculation of the fitted lines in Figure 3a is exact for the model used, and there are no assumptions in the derivation of the area per molecule. In particular, the calculation uses only the overall scattering length of the surfactin, which is accurately determined from the isotopic composition; it does not require the use of the volume of the molecule, which is less certain. The error in the determination of the area per molecule was estimated using least-squares minimization and the procedure illustrated in Figure 5 below. Determinations of molecular area from the alternative method of surface tension and the Gibbs equation are fraught with difficulty for biomolecules because the bulk charge and state of aggregation below the cmc need to be known precisely in order to identify the prefactor that appears (29) Fragneto, G.; Li, Z. X.; Thomas, R. K.; Rennie, A. R.; Penfold, J. J. Colloid Interface Sci. 1996, 178, 531. (30) Penfold, J.; Thomas, R. K.; Simister, E.; Lee, E.; Rennie, A. J. Phys.: Condens. Matter 1990, 2, Sa411.
Aggregation of Surfactin
Figure 3. Observed (points) and fitted (lines) neutron reflectivity profiles for (a) perdeuterated surfactin (O), leucine deuterated surfactin (4), and hydrophile + chain deuterated surfactin (b), all in NRW, and (b) fully hydrogentated surfactin (1), perdeuterated surfactin (O), leucine deuterated surfactin (4), and hydrophile + chain deuterated surfactin (b), all in D2O. The concentration was 6 × 10-6 M, and the pH was 7.5. Each profile is shifted by -2 from the one above. The parameters for the calculated profiles are given in the text.
in the Gibbs equation and to make the conversion to area per adsorbed molecule. This neutron measurement is therefore the first direct model-independent measurement of the molecular area of a naturally occurring biosurfactant at the air/water interface. At this stage, it is interesting that the in-plane diameter of the molecule, at 14 Å taking r ) A/π, is similar to the normal dimension (i.e., the molecule appears to be approximately spherical, which is not how it is drawn in Figure 2). The second limiting contrast condition is to use D2O as the subphase. For this contrast, if the surfactin is in its protonated form, for which the scattering length density is not very different from that of air, then the reflected signal is mainly that of D2O from a surface layer from which D2O has been displaced to an extent that depends on how the surfactin embeds itself in the water. If the surfactin is not completely embedded in the water, then deuteration of the surfactin can lead to further contributions to the reflectivity from the deuterated parts. To optimize the resolution of the structural determination, we measured profiles of the same three labeled surfactins used to obtain the NRW data and of fully protonated surfactin, all in D2O, and these are shown in Figure 3b. The modeling of these profiles is more complicated than for the NRW profiles. We use the method of partial structure factors (eq 1) in which the reflectivity can be modeled in terms of separate contributions from the labeled fragments of the surfactin, each described as a Gaussian distribution along the surface normal (eq 4) and a contribution from a water layer that fills the available space up to a certain level in the surface, after which it decays according to a half-Gaussian. The fragments were taken to be the four leucines combined into one group, the hydrocarbon chain, and the remainder of the ring, which we will refer to as the hydrophile because it contains the two charged residues. The amount of each fragment is constrained by the known composition of the molecule. Unlike the determination of the area per molecule, described above, the volumes of the
Langmuir, Vol. 25, No. 7, 2009 4215
Figure 4. Distributions of adsorbed surfactin fragments and water along the direction normal to the air/water interface: (a) chains (green line), leucines (red line), hydrophiles (purple line), and water (blue line) and (b) total surfactin (red line), water (blue line), and total water and surfactin (green line). The parameters are those used to calculate the fitted lines in Figure 3.
fragments become important because they determine, for example, the displacement of water. The reflectivity can be calculated exactly for such a model and is found to be particularly sensitive to the separation of the fragments along the surface normal. The main assumptions are then the volumes for each fragment, which were estimated from fragments of known structures to be 670 (the four leucines), 375 (the hydrocarbon chain), and 475 Å3 (the hydrophile).20,21 The scattering length densities are known accurately from the composition of the fragments, and allowance was made for deuterium exchange of the exchangeable protons in the structure. The optimum fit of a single structure to the set of all seven profiles in Figure 3 (continuous lines) gives the following separations between fragments, where zero corresponds to the Gibbs plane for zero adsorption of water: chain-water 7 Å, hydrophile-water 1 Å, and leucines-water 6.5 Å, all (1 Å. The widths of the distributions, defined as above, were 9 ( 2 Å for the leucines and 13.5 ( 3 Å for the other fragments. The distributions of the fragments are plotted in Figure 4. The volume fraction of surfactin as a whole in the layer increases to very close to 1.0, indicating an unusually closely packed surface layer. The sensitivity of the fits of model structures to the reflectivity data is usually best explored by a systematic study of the variation of the different parameters. In Figure 5, we show the variation of the sum of the residuals as two sets of different types of parameters are varied: (a) the width of a distribution and (b) the separation between a pair of distributions. From these two plots, it can be seen that the method is indeed very sensitive to separations between fragments but less so to the widths of the distributions. The errors quoted above are based on these and equivalent plots. A possible structure that takes account of the neutron results is sketched in Figure 6. The important features are that the chain must be folded back into the leucines of the heptapeptide ring in order to give the observed compactness and the low extent of immersion in the aqueous subphase compared with that of
4216 Langmuir, Vol. 25, No. 7, 2009
Figure 5. (a, b) Sensitivity of the best fits to the data, as measured by the sum of the residual squares, to variations in (a) the width (σ) of the chain + hydrophile (b) and the leucine (O) distributions and (b) the hydrophile-leucines (O) and hydrophile-water (b) separations (with the latter displaced from the true separation in order to be plotted on the same scale).
Figure 6. Structural arrangement of the surfactin skeleton: C in the ring (black), C in the chain (gray), O (red), N (green), and O- (magenta). This is the same as in Figure 2 except that the chain has been folded over so that it is in contact with the leucines on one side of the molecule. This gives it dimensions that agree much more closely with those observed in the neutron reflection experiment than the structure in Figure 2.
synthetic surfactants.30,31 The structural determination described here goes well beyond the detail of any other experiments on surfactin, and there is little with which to compare. A major objective in the surface measurements and the computer simulations to date has been to determine the area occupied by the molecule at the saturated interface, A. Maget-Dana and Ptak used both surface tension and surface pressure-area curves to determine A.32 The application of the Gibbs equation to the surface tension gave a value of A of 132 Å2 for the ionized molecule at concentrations close to the cmc, somewhat lower than our direct experimental value of 147 Å2 but with the ever present uncertainty in the use of the Gibbs equation on multiply charged surfactants. (31) Lu, J. R.; Li, Z. X.; Thomas, R. K.; Penfold, J. J. Chem. Soc., Faraday Trans. 1996, 92, 403. (32) Maget-Dana, R.; Ptak, M. J. Colloid Interface Sci. 1992, 153, 285.
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Although Maget-Dana and Ptak measured the Π-A isotherm (i.e., treating the deposited surfactin layer as insoluble) at pH 7.4, they did not give an analysis of the results. From their plot, we estimate that the onset of pressure occurs over an area, A0, in the region of 250 Å2. The shape of the curve at high pressure is difficult to analyze and, indeed, does not reach the value of Π corresponding to the known limiting surface tension, indicating the loss of material by solubilization into the subphase. MagetDana and Ptak, Ishigami et al.,33 and Gallet et al.6 measured values of A0 and At (at the transition from expanded to solid surface phases) at low pH, where the surfactin is not ionized and the surface film is more stable. Their values range from 154 to 215 Å2 for A0 and from 89 to 132 Å2 (for the surfactin with a C14 chain) for At. Because our measurements were made at the cmc, where the surface pressure is about 40 mN m-1, our value of 147 Å2 should be compared with At. There should be some increase in At when the molecule ionizes, but our directly observed value is larger than the highest of the alternative values for At. The very low values sometimes observed by others have led to the suggestion that the peptide ring of surfactin orients normal to the surface under high compression. This is consistent neither with the neutron value of A nor with the structure obtained from neutron reflection. Better estimates of the area per surfactin come from computer simulations. Gallet et al. found a value of 153 Å2 (C14 side chain) with the side chain folded onto the leucines and the peptide ring in the S2 conformation. However, Gallet et al.’s values of A for the extended and folded conformations with either S1 or S2 peptide rings all cluster in the range of 137-153 Å2, which are in reasonable agreement with the neutron result except that the simulation was done on the uncharged molecule. Previous discussions of the surface arrangement of surfactin have been based on values of A that have been determined indirectly and have therefore been uncertain. Our structural determination shows clearly that (i) the hydrocarbon chain must fold back into the structure in some way, presumably to associate itself with the hydrophobic leucines (or valine), (ii) the overall thickness of the layer is such that the peptide ring must be aligned parallel with the surface, even at the high surface pressure of the experiment, and (iii) the separation of the hydrophile is 5.5 Å from its relatively large hydrophobic neighbor (center-to-center distance). It is useful to compare these conclusions with the results of the two computer simulations, although again these were done on the neutral molecule. Gallet et al. concluded that surfactin adsorbed with the plane of the peptide ring aligned with the surface and that the most stable structure at the interface was one with the chain folded back so as to interact with leucine 2 and valine 4. Although the neutron results do not indicate with which residues the chain interacts, they show unequivocally that it must interact strongly with the side chains of the hydrophobic amino acids. Gallet et al. also concluded that the S2 conformation of the peptide ring was preferred at the interface and that the molecular area for this conformation with folded chain was 153 Å2. Nicolas’ molecular dynamics simulation of surfactin at an oil/water interface was made at a number of fixed areas per molecule, which makes it more difficult to compare.14 There is nevertheless indirect agreement with our result (and Gallet et al.’s) that the surfactin forms a very compact unit in that Nicolas found a surprisingly high rate of tumbling of the molecule in his simulation (i.e., the hydrophilic group spends a significant amount of time pointing upward into the oil phase and is not distinct from the rest of the molecule). Given also that the head groups (33) Ishigami, Y.; Osman, M.; Nakahara, H.; Sano, Y.; Ishiguro, R.; Matsumoto, M. Colloids Surf., B 1995, 4, 341.
Aggregation of Surfactin
in surfactin are much smaller relative to the hydrophobic groups than in most conventional synthetic surfactants, the distribution of material in adsorbed surfactin more closely resembles a hydrophobic sphere (or ellipsoid) with two charges on the underside than a conventional surfactant with well-separated hydrophobic and hydrophilic units. This is clearly shown in the distributions of Figure 4 (examples of the corresponding types of distribution of conventional surfactants can be found in refs 26 and 34) and is represented diagramatically in Figure 6, which is based on the exact coordinates of Bonmatin et al.’s S2 conformation of the peptide ring5 and a plausible folded back conformation of the chain toward one pair of leucines (the four leucines lie approximately in a tetrahedral arrangement). The aqueous subphase does not penetrate very far into this structure in that the center of the distribution of the hydrophile is more than 1 Å above the Gibbs plane defining the surface excess of water as zero. Surfactin Adsorbed at the Hydrophobic Solid/Aqueous Interface. The air/water interface is a weakly hydrophobic/ aqueous interface, and it is interesting to see how the structure and packing of surfactin changes at a strongly hydrophobic/ aqueous interface. The very compact nature of the molecule at the air/water interface suggests that it ought to change very little at the more hydrophobic surface. We therefore explored the adsorption of surfactin at the strongly hydrophobic surface formed by a self-assembled monolayer of octadecyl trichlorosilane (OTS). OTS monolayers have been investigated at the air-solid interface by many techniques such as ellipsometry,35 infrared spectroscopy,36 X-ray reflection,37 atomic force microscopy,38,39 and contact angle measurements.40 Fragneto et al.29 have used neutron reflectometry to characterize the OTS monolayer under water, using perdeuterated OTS (d-OTS) and to examine the adsorption of surfactants at this layer. We followed the procedures in that paper. The reason for using a d-OTS surface is that it creates a strong contrast between any adsorbed layer on the OTS and the OTS. As a result, a fringe is often observed whose minimum defines the combined OTS + adsorbed layer thickness independently of the composition, which is not the case for the air/water interface. The difference between the reflectivity at the two surfaces results from the much greater total thickness of the combined layer in the OTS system. Because the thickness of the OTS itself can also be determined with good accuracy, the method provides an independent determination of the thickess of the surfactin layer. The d-OTS layer was characterized with three different water contrasts: H2O, D2O, and water matched to silicon following the procedures described by Fragneto et al. Following this characterization, the adsorption of fully protonated surfactin in D2O at its cmc was measured. In this situation, the surfactin gives a strong signal as shown in Figure 7, indicating strong adsorption. Its area and dimension normal to the surface can be determined from this data with higher precision than at the air/water interface for the reason given above. The best fit to the data was obtained using the optical matrix model for calculating the reflectivity with three uniform sublayerssone each for the SiO2 layer on the silicon, the d-OTS, and the surfactinswith the parameters of the (34) Li, Z. X.; Dong, C. C.; Wang, J. B.; Thomas, R. K.; Penfold, J. Langmuir 2002, 18, 6614. (35) Fujii, M.; Sugisawa, S.; Fukada, K.; Kato, T., T.; Shirakawa; Seimiya, T. Langmuir 1994, 10, 984. (36) Hoffmann, H.; Mayer, U.; Krischanitz, A. Langmuir 1995, 11, 1304. (37) Daillant, J.; Benattar, J.; Leger, L. Phys. ReV. A 1990, 41, 1963. (38) Bierbaum, K.; Grunze, M. Langmuir 1995, 11, 2143. (39) Nakagawa, T.; Ogawa, K.; Kurumizawa, T. J. Vac. Sci. Technol., B 1994, 12, 2215. (40) Horr, T.; Ralston, J.; Smart, R. Colloids Surf. 1995, 97, 183.
Langmuir, Vol. 25, No. 7, 2009 4217
Figure 7. Observed (points) and fitted (lines) neutron reflectivity profiles for surfactin adsorbed at its cmc (6 × 10-6 M) and at pH 7.5 in D2O on a hydrophobic self-assembled monolayer of fully deuterated octadecyl trichlorosilane (b). Open circles represent the date for the OTS layer in the absence of surfactin. The surfactin data was fitted with a single uniform layer of thickness 15 ( 1 Å and an area per molecule of 145 ( 5 Å2. The error bars are comparable to the size of the points.
silica and OTS layers being fixed by the preliminary characterization. At this contrast, the reflectivity would be sensitive to any penetration of the outer part of the perdeuterated OTS by the protonated surfactin, but no such effect was observed. At this hydrophobic surface, the area per surfactin is 145 ( 5 Å2, within error the same as that at air/water, and the thickness is 15 ( 1 Å, using the same procedure for estimating the errors as described above. This thickness is defined in terms of a uniform layer and is not obviously comparable to the 14 Å for the surfactin at the air/water interface, which is based on the distribution of eq 4. However, it has been shown that in practice the σ defined by eq 4 is much closer to the effective uniform layer thickness than the true half-width of the equivalent Gaussian.26 The close resemblance of the two values of the thickness therefore indicates that the conformation of the surfactin is the same at the two interfaces. The expectation that amphiphilic molecules should reach their maximum packing on a hydrophobic surface and the resemblance of the area per molecule at the two surfaces suggest that surfactin has also reached its packing limit at the air/water interface. For a synthetic surfactant, the packing is generally significantly tighter at the more hydrophobic surface, and there are often associated changes in structure. Small-Angle Neutron Scattering. The representation of Figure 6 is different from the most commonly assumed conformation of surfactin, which is closer to that shown in Figure 2. The biological role of surfactin is not known, and it may be that this difference in structure is important when one tries to assess surfactin’s biological role. The shape of the aggregates formed by a surfactant in solution is determined by the geometrical packing parameter (V/Al, where V is the volume of the hydrophobic unit, A is the area of the headgroup, and l is the fully extended length of the molecule). Using the estimates of the volumes of the constituent fragments given earlier in conjunction with the measured neutron parameters leads to two estimates of the geometrical packing parameter, depending on what one takes as the hydrophobic unit. Taking the neutron view that the leucines and chain form the hydrophobic unit (as shown in Figure 6) gives a value 0.65, whereas the division into chain as hydrophobe and peptide ring as hydrophile (Figure 2) gives 0.21. The latter would mean that micelles were the preferred type of aggregate, and the former would favor a lamellar structure (e.g., vesicles). To examine this issue further, we have determined the structure of the solution aggregates using neutron small-angle scattering (SANS) at the same pH of 7.5.
4218 Langmuir, Vol. 25, No. 7, 2009
Figure 8. Observed (points) and fitted (lines) small-angle neutron scattering patterns for fully protonated surfactin (O) and surfactin with a perdeuterated chain and hydrophile and protonated leucines (b) in D2O. The concentration was 1 mM, and the pH was 7.5. The fitted lines are for micelles with an aggregation number of 20 with a core radius of 22 Å containing the chains and leucine groups and an overall radius of 25 Å.
The SANS results for the fully protonated surfactin and the deuterated leucine version both in D2O at 1 mM are shown in Figure 8. The data were modeled using a core-shell model form factor with geometrical constraints and constrained by the absolute cross section and the solution concentration. The only satisfactory fits of a core-shell model, using eq 2 with spherical form factors for core and shell, were obtained when the core contains all of the chain group and the four leucines and the shell contains the remaining headgroup, water, and the counterions. These fits are shown as continuous lines in Figure 8. The geometrical parameters of the micelle were found to be strongly constrained by the surfactin aggregation number and respective fragment volumes. Fits to the two sets of data using the same fragment volumes as for the reflectivity show that the aggregation number at this pH is unusually small at 20 ( 4 with an overall micelle diameter of 50 ( 5 Å and a hydrophobic core radius of 22 ( 2 Å. The cross sections in Figure 8 are very small, and this is shown by the inaccuracies in the backgound subtraction for the weaker of the two curves. This is a different result from that obtained under more or less comparable conditions using electron cryomicroscopy by Knoblich et al.41 These authors found a distribution of micelle shapes from spherical to ellipsoidal with diameters in the range of 50-90 Å and lengths of up to 190 Å that mostly lie outside our range of error. At first sight, the presence of micelles supports the view that the whole peptide ring ought to be thought of as the hydrophilic unit. However, the two sets of data in Figure 8 can be fit only with a self-consistent model when the leucines are in the hydrophobic core of the micelle. Surfactin is therefore a molecule whose behavior is not easily described in terms of a packing parameter, and this is also reflected in several of its properties. Thus, there is strong evidence that surfactin is a powerful foam and emulsion stabilizer, a property that correlates better with the higher value of the packing parameter suggested from neutron reflectometry than the lower value suggested by the SANS result. (41) Knoblich, A.; Matsumoto, M.; Ishiguro, R.; Murata, K.; Fujiyoshi, Y.; Ishigami, Y.; Osman, M. Colloids Surf., B 1995, 5, 43.
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The concept of a packing parameter is evidently difficult to apply with confidence to a molecule of this shape, so it may be more helpful to think of the surface properties of surfactin in terms of the factors that stabilize Pickering emulsions.42 These widely occurring emulsions are stabilized by particles adsorbing at the oil/water interface, and the stabilization energy comes from the replacement of the high-energy oil/water interface by particle/ water and particle/oil interfaces. The direction of stabilization (oil in water or water in oil) depends on which of these two energies is the lower. Surfactin could be regarded as a sphere with one side preferring oil and the other favoring water. The objection to using such an approach is that the advantages of Pickering-type stabilization vanish when the rate of exchange of particle or surfactant between bulk and surface becomes fast as the particle becomes too small.43 However, at the very low cmc of surfactin the rate of exchange with the surface may be sufficiently low for the Pickering concept to be more useful than that of the packing parameter. Thermodynamics generally restricts the range of possibilities for manipulating conventional smallmolecule surfactant architecture to optimize adsorption at the expense of aggregation. If the biological needs are for adsorption, then aggregation becomes an expensive waste, and the unusual design of surfactin, which favors adsorption while keeping the aggregation number low, unlike conventional small-molecule surfactants, suggests that nature’s objective with the surfactin molecule is surface activity rather than aggregation.
Conclusions Nature produces a large number of biosurfactants, many of which have interesting potential applications either as more sustainable substitutes for what we already use or in medicine. An important first step in applying them in either area is to understand how and why such molecules aggregate either in the bulk or at surfaces. The latter is very challenging in that there is a general weakness in experimental techniques capable of simultaneously probing the composition and structure of wet, disordered interfaces. We have used selective deuteration in conjunction with neutron reflectometry to investigate the structure and surface density of the biosurfactant, surfactin, at two types of surface, and we have also examined its bulk aggregation. This is the first study of a naturally occurring biosurfactant and the compact surface structure of surfactin with results that differ somewhat from the standard division of synthetic surfactants into a separate hydrophile and hydrophobe, suggesting that the study of such molecules could lead to new ideas for the design of synthetic surfactants. Acknowledgment. H.-H.S. thanks the Swire Foundation for a scholarship. We also thank Dr. I. Grillo at Institut LaueLangevin, Grenoble, France, for assistance with the D11 experiment, Drs. D. W. Hsu and C. Pears at the Department of Biochemistry, University of Oxford, for help with the preparation and purification of surfactin, and Professor J. Bonmatin for kindly sending us his NMR coordinates for the heptapeptide ring. LA802913X (42) Aveyard, R.; Binks, B. P.; Clint, J. H. AdV. Colloid Interface Sci. 2003, 100, 503. (43) Martin, L. F.; Fieser, E. L. Org. Synth. 1943, 2, 560.
