ABSTRACT Surfactin is a bacterial lipopeptide with powerful surfactant-like properties. High-sensitivity isothermal titration calorimetry was used to study the self association and membrane partitioning of surfactin. The critical micellar concentration (CIVIC), was 7.5 (mu)M, the heat of micellization was endothermic with delta(H)^sup w->m^^sub Su^ = +4.0 kcal/mol, and the free energy of micellization delta(G)^sup O,w->m^^sub Su^ - -9.3 kcal/mol (25degC; 100 mM NaCI; 10 mM TRIS, 1 mM EDTA; pH 8.5). The specific heat capacity of micellization was deduced from temperature dependence of delta(H)^sup w->m^^sub Su^ as delta(C)^sup w->m^^sub p^ = -250 +/- 10 cal/(mol*k). The data can be explained by combining the hydrophobicity of the fatty acyl chain with that of the hydrophobic amino acids. The membrane partition equilibrium was studied using small (30 nm) and large (100 nm) unilamellar POPC vesicles. At 25*C, the partition coefficient, K, was (2.2 =/- 0.2) x 10^sup 4^ M^sup -1^ for large vesicles leading to a free energy of delta(G)^sup O,w->b^^sub Su^ = -8.3 kcal/mol. The partition enthalpy, K, was again endothermic, with delta(H)^sup w->b^sub Su^ = 9 +/- 1 kcal/mol. The strong preference of surfactin and a varity of non-ionic membrane insertion explains the high membrane-destabilizing activity of the peptide. For surfactin and a variety of non-ionic detergents, the surfactant-to-lipid ratio, inducing membrane solubilization, R^sup sat^^sub b^, can be predicted by the simple relationship R^sup sat^^sub b^ ~/= K * CMC.
The growing resistance of bacteria against conventional antibiotics has led to an intense search for new types of antibiotics such as antibiotic peptides. Among these, surfactin is a detergent-like lipopeptide produced by Bacillus subtilis (Arima et al., 1968) reducing the surface tension of water from 72 mN/m to ~30 mN/m at concentrations of ~10 (mu)M (Ishigami et al., 1995; Peypoux et al., 1999). Surfactin consists of a heptapeptide headgroup with the sequence Glu-Leu-D-Leu-Val-Asp-D-Leu-Leu closed to a lactone ring by a C^sub 14-15^ beta-hydroxy fatty acid. The peptide ring adopts a "horse-saddle" structure in solution with the two charged residues forming a "claw," which is a potential binding site for divalent cations (Bonmatin et al., 1992). On the opposite side of the ring, the fatty acyl chain may extend into a micellar structure or into a lipid bilayer. Surfactin has a critical micellar concentration (CMC) of 9.4 taM in 200 mM NaHC.03 at pH 8.7 (Ishigami et al., 1995) and forms rod-like micelles with an aggregation number of ~ 170. The pK^sub a^ of aggregated surfactin is about 6 (Maget-Dana et al., 1992).
A variety of important applications and physiological activities have been proposed for surfactin. Surfactin could play a physiological role by increasing the bioavailability of water-insoluble substrates and by regulating the attachment/ detachment of microorganisms to and from surfaces (Rosenberg and Ron, 1999). Surfactin has hemolytic (Kracht et al., 1999), antiviral (Vollenbroich et al., 1997a; Kracht et al., 1999), antibacterial (Vollenbroich et al., 1997b; Beven and Wroblewski, 1997), and antitumor (Kameda et al., 1974) properties. These observations have attracted considerable interest because they may all be related to the effect of surfactin on the lipid part of the biological membrane. The application of surfactin as a strong, biodegradable detergent for technical and household purposes can also be envisaged but would require much cheaper production methods (Rosenberg and Ron, 1999).
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Heiko Heerklotz and Joachim Seelig
Department of Biophysical Chemistry, Biocenter of the University of Basel, CH-4056 Basel, Switzerland
Received for publication 12 March 2001 and in final form 31 May 2001.
Address reprint requests to Joachim Seelig, Dept. of Biophysical Chemistry, Klingelbergstr. 70, Univ. of Basel-Biocenter, CH-4056 Basel, Switzerland. Tel.: 41-61-267-2190; Fax: 41-61-267-2189; E-mail: joachim. firstname.lastname@example.org.
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