VATER association

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.

INTRODUCTION

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).

REFERENCES

Arima, K., A. Kakinuma, and G. Tamura. 1968. Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem. Biophys. Res. Commun. 31:488-494.

Baker, B. M., and K. P. Murphy. 1998. Prediction of binding energetics from structure using empirical parametrization. Methods Enzymol. 295: 294-314.

Beschiaschvili, G., and J. Seelig. 1992. Peptide binding to lipid bilayers. Nonclassical hydrophobic effect and membrane-induced pK shifts. Biochemistry. 31:10044-10053.

Beven, L., and H. Wroblewski. 1997. Effect of natural amphipathic peptides on viability, membrane potential, cell shape and motility of mollicutes. Res. Microbiol 148:163-175.

Binford, J. S., Jr., and I. Wadso. 1984. Calorimetric determination of the partition coefficient for chlorpromazine hydrochloride in aqueous suspensions of dimyristoylphosphatidylcholine vesicles. J. Biochem. Biophys. Methods. 9:121-131.

Bonmatin, J. M., M. Genest, M. C. Petit, E. Gincel, J. P. Simorre, B. Comet, X. Gallet, A. Caille, H. Labbe, F. Vovelle, and M. Ptak. 1992. Progress in multidimensional NMR investigations of peptide and protein 3-D structures in solution. From structure to functional aspects. Biochimie. 74:825-836.

Chellani, M. 1999. Isothermal titration calorimetry: biological applications. Amer. Biotechnol. Lab. 17:14-18.

Clint, J. H. 1992. Surfactant Aggregation. Blackie & Son, Glasgow, U.K. 115.

Cooper, A. 2000. Heat capacity of hydrogen-bonded networks: an alternative view of protein folding thermodynamics. Biophys. Chem. 85: 25-39.

Emerson, M. F., and A. Holtzer. 1965. On the ionic strength dependence of micelle number. J Phys. Chem. 69:3718-3721.

Gazzara, J. A., M. C. Phillips, S. Lund-Katz, M. N. Palgunachari, J. P. Segrest, G. M. Anantharamaiah, W. V. Rodrigueza, and J. W. Snow. 1997. Effect of vesicle size on their interaction with class A amphipathic helical peptides. J. Lipid Res. 38:2147-2154.

Heerklotz, H., and R. M. Epand. 2001. The enthalpy of acyl chain packing and the apparent water accessible apolar surface area of phospholipids. Biophys. J. 80:271-279.

Heerklotz, H., and J. Seelig. 2000a. Correlation of membrane/water partition coefficients of detergents with the critical micelle concentration. Biophys. J. 78:2435-2440.

Heerklotz, H., and J. Seelig. 2000b. Titration calorimetry of surfactant-- membrane partitioning and membrane solubilization. Biochim. Biophys. Acta. 1508:69-85.

Hiemenz, P. C. 1986. Principles of colloid and surface chemistry. Marcel Dekker Inc., New York.

Ishigami, Y., M. Osman, H. Nakahara, Y. Sano, R. Ishiguro, and M. Matsumoto. 1995. Significance of beta-sheet formation for micellization and surface adsorption of surfactin. Colloids Surf. B. 4:341-348.

Kameda, Y., S. Oira, K. Matsui, S. Kanatomo, and T. Hase. 1974. Antitumor activity of Bacillus natto. V. Isolation and characterization of surfactin in the culture medium of Bacillus natto KMD 2311. Chem. Pharm. Bull. (Tokyo). 22:938-944.

Kracht, M., H. Rokos, M. Ozel, M. Kowall, G. Pauli, and J. Vater. 1999. Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives. J. Antibiot. (Tokyo). 52:613-619.

Kresheck, G. C. 1998. Comparison of the calorimetric and van't Hoff enthalpy of micelle formation for a nonionic surfactant in H20 and D20 solutions from 15 to 40degC. J. Phys. Chem. B. 102:6596-6600.

Kresheck, G. C., and W. A. Hargraves. 1974. Thermometric titration studies of the effect of head group, chain length, solvent, and temperature on the thermodynamics of micelle formation. J. Coll. Interf Sci. 48:481-493.

Lasch, J. 1995. Interaction of detergents with lipid vesicles. Biochim. Biophys. Acta. 1241:269-292.

MacDonald, R. C., R. I. MacDonald, B. P. Menco, K. Takeshita, N. K. Subbarao, and L. R. Hu. 1991. Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim. Biophys. Acta. 1061: 297-303.

Maget-Dana, R., and M. Ptak. 1992. Interfacial properties of surfactin. J. Coll. Interf Sci. 153:285-291.

Maget-Dana, R., L. Thimon, F. Peypoux, and M. Ptak. 1992. Surfactin/ iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A. Biochimie. 74:1047-1051.

Matsuzaki, K., and J. Seelig. 1994. NMR study on interactions of an antibiotic peptide, magainin-2, with lipid bilayers. In Peptide Chemistry. N. Ohno, editor. Protein Research Foundation, Osaka, Japan. 129-132.

Muller, N. 1990. Search for a realistic view of hydrophobic effects. Acc. Chem. Res. 23:23-28.

Muller, N. 1992. Does hydrophobic hydration destabilize protein native structures? TIBS. 17:459-463.

Olofsson, G. 1985. Microtitration calorimetric study of the micellization of three poly (oxyethylene) glycol dodecyl ethers. J. Phys. Chem. 89: 1473-1477.

Paula, S., W. Sus, J. Tuchtenhagen, and A. Blume. 1995. Thermodynamics of micelle formation as a function of temperature: a high sensitivity titration calorimetry study. J. Phys. Chem. 99:11742-11751.

Peypoux, F., J. M. Bonmatin, and J. Wallach. 1999. Recent trends in the biochemistry of surfactin. AppL Microbiol. Biotechnol. 51:553-563. Privalov, P. L., and S. J. Gill. 1989. The hydrophobic effect: a reappraisal. Pure Appl. Chem. 61:1097-1104.

Rosenberg, E., and E. Z. Ron. 1999. High- and low-molecular-mass microbial surfactants. Appl. Microbiol. Biotechnol. 52:154-162.

Seelig, J. 1997. Titration calorimetry of lipid-peptide interactions. Biochim. Biophys. Acta. 1331:103-116.

Seelig, J., and P. Ganz. 1991. Nonclassical hydrophobic effect in membrane binding equilibria. Biochemistry. 30:9354-9359.

Spolar, R. S., J. R. Livingstone, and M. T. Record, Jr. 1992. Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water. Biochemistry. 31:3947-3955.

Spuhler, P., G. M. Anantharamaiah, J. P. Segrest, and J. Seelig. 1994. Binding of apolipoprotein A-I model peptides to lipid bilayers. Measurement of binding isotherms and peptide-lipid headgroup interactions. J. Biol. Chem. 269:23904-23910.

Tanford, C. 1980. The Hydrophobic Effect: Formation of Micelles and Biological Membranes. Wiley, New York. 61-77.

Vollenbroich, D., M. Ozel, and J. Vater, R. M. Kamp, and G. Pauli. 1997a. Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis. Biologicals. 25:289-297.

Vollenbroich, D., G. Pauli, M. Ozel, and J. Vater. 1997b. Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis. Appl. Environ. Microbiol. 63:44-49.

Wenk, M. R., T. Alt, A. Seelig, and J. Seelig. 1997. Octyl-beta-D-- glucopyranoside partitioning into lipid bilayers: thermodynamics of binding and structural changes of the bilayer. Biophys. J. 72:1719-1731.

Wieprecht, T., 0. Apostolov, M. Beyermann, and J. Seelig. 2000a. Membrane binding and pore formation of the antibacterial peptide PGLa: thermodynamic and mechanistic aspects. Biochemistry. 39:442-452.

Wieprecht, T., 0. Apostolov, and J. Seelig. 2000b. Binding of the antibacterial peptide magainin 2 amide to small and large unilamellar vesicles. Biophys. Chem. 85:187-198.

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. seelig@unibas.ch.

Copyright Biophysical Society Sep 2001
Provided by ProQuest Information and Learning Company. All rights Reserved