Journal of Peptide Science
`J. Peptide Sci. 4: 449–458 (1998)
`
`Tuning Micelles of a Bioactive Heptapeptide Biosurfactant
`via Extrinsically Induced Conformational Transition of
`Surfactin Assembly
`
`MOHAMAD OSMANa,*, HARALD HØILANDb, HOLM HOLMSENc and YUTAKA ISHIGAMId
`
`a School of Science and Technology (HIS), Ullandhaug, Stavanger, Norway
`b Department of Chemistry, University of Bergen, Bergen, Norway
`c Department of Biochemistry and Molecular Biology, University of Bergen, Bergen, Norway
`d National Institute of Materials and Chemical Research, Tsukuba, Ibaraki, Japan
`
`Received 16 September 1997
`Accepted 31 March 1998
`
`Abstract: We have studied the effects of extrinsic environmental conditions on the conformation of surfactin,
`a heptapeptide biosurfactant from Bacillus subtilis, in aqueous solutions. It has been made clear that
`temperature, pH, Ca2(cid:27) ions and the synthetic nonionic surfactant hepta-ethylene glycol (C12E7) affect the
`conformation of surfactin in aqueous solutions. The b-sheet formation reached a maximum at 40°C both in
`presence and absence of (C12E7) and the nonionic surfactant enhances the b-sheet formation even at 25°C.
`Ca2 (cid:27) induced the formation of a-helices and caused this transition at 0.3 mM with surfactin monomers or
`at 0.5 mM with surfactin micelles, but above these transition concentrations of Ca2(cid:27) b-sheets were
`observed. In micellar solution the b-sheet structure was stabilized at pH values below 7 or upon addition
`of Ca2(cid:27) in concentrations above 0.5 mM. Our results indicated that the bioactive conformation of surfactin
`is most likely the b-sheets when the molecules are assembled in micelles. The b-sheet structure in micelles
`could be retained by tuning the micelles. Surfactin micelles could be tuned in the bioactive conformation by
`manipulating pH, temperature, Ca2(cid:27) or (C12E7) concentrations in surfactin solutions. Our results strongly
`indicated that Ca2(cid:27) and other molecules (such as C12E7) may function as directing templates in the
`assembly and conformation of surfactin in micelles. Thus, we suggest environmental manipulation and
`template-aided micellation (TAM) as a new approach for preparing predesigned micelles, microemulsions or
`micro-spheres for specific application purposes. © 1998 European Peptide Society and John Wiley & Sons,
`Ltd.
`
`Keywords: peptide surfactant; surfactin; conformation; micelles; biosurfactant
`
`INTRODUCTION
`
`Surfactin is a lipopeptide produced by Bacillus sub-
`tilis [1–7] and is abundant in many natural prod-
`ucts such as natto (fermented soy beans) [5,8–11].
`It was fist produced and characterized by Kakinuma
`et al. [1–3]. Since, many analogues have been pro-
`duced and studied [10–14]. Surfactin is a biosur-
`
`Abbreviations: Sf, surfactin; C12E7, heptaethylene glycol.
`
`* Correspondence to: School of Science and Technology (HIS), P.O.
`Box 2557, Ullandhaug, N-4004 Stavanger, Norway.
`
`© 1998 European Peptide Society and John Wiley & Sons, Ltd.
`CCC 1075–2617:98:070449-10$17.50
`
`factant with a high industrial and commercial
`potential because of its superb surface and interfa-
`cial activity [1,2,15] and because it has a diversity of
`bioactive properties [1–3,8–11,16–22].
`It
`forms
`large micelles at a very low concentration [23] and
`the cmc of the different analogues is of the order
`10(cid:28) 5 M [4,21,23] or less. Surfactin has also shown
`ionophoric and sequestering properties [21,24]. The
`bioactivities of surfactin include: inhibition of blood
`clotting [1–3], haemolytic activity [21], repression of
`cAMP phosphodiesterase [14,16–18], hypocholes-
`terolemic action [22], channel formation in mem-
`branes [25], synergistic antifungal activity when
`
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`OSMAN ET AL.
`
`combined with iturins [19,20], antibiotic activity
`[3,26], antitumour action [8,9] and anti HIV effects
`[11]. These physicochemical and biological activities
`are most likely related to the mode of molecular
`assembly of surfactin in micelles as well as the
`secondary structure and the conformation of the
`surfactin molecules in the aggregates. The relation
`of the conformation of peptides and their physico-
`chemical properties to their biological actions have
`been reported by many authors [27–33]. It has also
`been observed that peptides adopt multiple confor-
`mation in different environments or under distinct
`conditions in the milieu [34–37].
`The peptide conformation is not directed solely by
`the primary structure of the peptide or its intrinsic
`properties but it is also controlled by the con-
`stituents of the extrinsic environment [34–45]. Pep-
`tide may form ion channels, proteins or undergo
`conformational transitions depending on extrinsic
`templates [39], ion types [38,44], hydrophobicity:hy-
`drophilicity of the environment [43], redox proper-
`ties of
`the environment
`[40],
`types of applied
`solvents [37,42] influence of pH [4] and temperature
`[45]. The importance of the conformation of peptides
`in general and the peptide-biosurfactant systems in
`particular is due to the relation of the conformation
`to the system stability as well as the specificity and
`ability of the peptides to bind to either ligands or
`receptors, and the resulting induced biological ac-
`tions. Although many reports have addressed these
`questions, there are no reports regarding the influ-
`ence of such environmental factors on the secondary
`structure, the conformation and the bioactivity of
`micelles formed by peptide biosurfactants. The in-
`vestigation of the conformation of lipopeptides, par-
`ticularly of peptide biosurfactants,
`is of crucial
`importance for the understanding of their potential
`applications. Systems based on biosurfactants have
`been suggested for use in drug targeting, controlled
`drug release, DDS (drug delivery systems), intelli-
`gent liposomes, transdermal absorbtion treatments
`and biosensors [46]. The effectiveness of such sys-
`tems may depend totally or partly on the correct
`conformation of the peptide biosurfactant molecules
`in the surfactant systems, such as liposomes, mi-
`celles, microemulsions
`and
`lipopeptide-micro-
`spheres. Therefore understanding the behaviour
`and the structural transitions in relation to the
`bioactive conformation of the lipopeptides in these
`surfactant systems, is crucial for designing suitable,
`stable and effective peptide biosurfactant systems.
`We have recently reported on the transition of
`a-helix structure to b-sheets in linear surfactin [47]
`
`and we have also reported on the formation of large
`micelles of cyclic surfactin by b-sheet formation
`[23].
`In the present paper we present our results re-
`garding the effects of the manipulation of the extrin-
`sic environment on the conformation of surfactin
`molecules and we discuss the stabilization of the
`secondary structures as well as the induction of
`conformational transitions which are favoured for
`bioactivity. We finally show the possibility of using
`this approach to tune micelles or prepare other
`micro-structures in surfactant systems of peptide
`biosurfactants where stability and biological efficacy
`can be retained at an optimum level.
`
`MATERIALS AND METHODS
`
`Surfactin was purchased from Wako Pure Chem
`Industries Ltd., Japan. The chemical structure of
`cyclic surfactin samples was ascertained by fast
`atom bombardment mass spectroscopy (FAB-MS)
`reported elsewhere [47]. The base peak, 1036.8 cor-
`responded exactly to the molecular weight of cyclic
`surfactin. The cyclic structure was further con-
`firmed by Furier-transformed Infrared spectroscopic
`(FTIR) measurements, the 1730 cm (cid:28) 1 band indi-
`cated the cyclic structure of the cyclic surfactin.
`All other chemicals were of reagent grade. The
`water used in the measurements was purified by
`passing through an ion exchange resin column,
`followed by distillation in an all quartz distillation
`column filled by tipped glass tubes.
`Phosphate buffered saline (PBS buffer) had the
`following formulation: NaCl 8.0 g, KH2PO4 0.2 g,
`Na2HPO4 1.15 g, KCl 0.2 g, per litre of distilled
`water. The pH was adjusted by 0.1 N HCl or 0.1 N
`NaOH to the desired pH.
`FAB mass spectra were taken by a JEOL HX-100
`double focusing mass spectrometer operated at a
`resolving power of 2000 (10% valley definition).
`About 1 mg of sample was dissolved in 2 ml of
`glycerol:H2O mixture (glycerol:H2O(cid:30)1:10 v:v) on a
`stainless-steel plate. A small amount of 1 N HCl was
`added for the measurement of positive ion spectra.
`Samples were bombarded with a xenon atomic beam
`of 6 keV. Data acquisition and processing were
`performed using JEOL JMADA 5000 system.
`FT-JR spectroscopy was performed on surfactin
`using KBr disks. The FT-JR spectra were measured
`using a JASCO FT-IR-5000 spectrophotometer and
`data were acquired and processed using JASCO-
`5000 software, version 2.2.
`
`© 1998 European Peptide Society and John Wiley & Sons, Ltd.
`
`J. Peptide Sci. 4: 449–458 (1998)
`
`PETITIONERS
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`EXHIBIT NO. 1017 Page 2 of 10
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`

`

`The CD spectra were measured using a JASCO J
`600 polarimeter and the data were acquired and
`processed using the J-600 software, version 2a. The
`spectra obtained were reasonably reproducible by
`repeated measurement under the forced experimen-
`tal conditions.
`The effect of temperature on the conformation of
`surfactin micelles both in the presence and absence
`of
`the nonionic surfactant Heptaethylene glycol
`(C12E7) were performed in 0.1 M NaHCO3 solutions
`having a pH of 8.7. In absence of C12E7 the sur-
`factin concentrations ranged from 1 to 3 mM and in
`the presence of C12E7 the surfactin concentrations
`ranged from 0.25 to 1 mM.
`The effect of temperature and C12E7 addition on
`weight-average (Mw) of surfactin micelles and the
`aggregation number was obtained in the manner
`described elsewhere [23].
`The CD spectra were obtained under strictly con-
`trolled temperatures in the range of 90.1°C. The
`CD spectra were taken in the temperatures range
`25–55°C. The effect of C12E7 on the conformation at
`different temperatures was measured in surfactin
`solutions having a concentration in the range 0.25–
`1 mM and the ratios of surfactin:C12E7 were either
`50:50 or 25:75.
`The molar elipticity (u) values whenever necessary
`were obtained directly from the CD spectra pro-
`cessed by the computer using the software men-
`tioned above.
`Effects of pH on the conformation were performed
`on a series of surfactin solutions in PBS buffer
`where pH values ranged from 6 to 9. A concentra-
`tion of 1(cid:29)l0(cid:28) 6 M, which is 1:10 of surfactin’s cmc,
`was used to examine the effects of pH on the confor-
`mation of surfactin in monomeric form. A concen-
`tration of 2(cid:29)10(cid:28) 5 M, which is twice the cmc of
`surfactin, was used to examine the effects of pH on
`the conformation of surfactin molecules in the mi-
`cellar form. Effects of pH on conformation were
`measured at 25°C.
`The effect of Ca2(cid:27) on the conformation was per-
`formed on a series of surfactin solutions in Tris
`buffer having a pH of 7.5. The concentrations of
`Ca2(cid:27) ranged from 0.1 0.7 mM.
`
`RESULTS AND DISCUSSION
`
`b-Turns and the Secondary Structure of Cyclic
`Peptides
`
`Cyclic peptides have been often used as a model
`
`TUNING MICELLES OF A PEPTIDE BIOSURFACTANT
`
`451
`
`studying b-turn conformation [48]. Since
`for
`surfactin is a cyclic peptide and CD spectra given
`later on may be confused with b-turn configura-
`tion, we do feel it necessary to address the ques-
`tion of b-turn. The b-turn configuration is also
`an important constituent of many proteins, where
`the polypeptide chain abides a relatively reversal
`turn in direction [49,50]. The spectrum of most
`common b-turn structures have a negative band
`near 225 nm, a very strong negative band at
`180–190 nm and a strong positive maximum at
`200–205 nm. There is noticeable difference of
`5–10 nm red shift between the maxima of b-turn
`and b-sheet spectra. However,
`in some special
`cases resemblance to a-helix have been observed,
`where the spectrum has negative bands near 220
`nm and 210 nm and a positive band near 190
`nm. The deviation in such cases is of few nano-
`metres and is not very distinct. The theoretical
`calculations have demonstrated that no single
`CD spectra could be assigned to the structural
`conformation called b-turn [51]. In cyclic peptides,
`the b-turn conformation may look like either a
`b-sheet or a-helix configuration. However, X-ray
`diffraction and NMR are used to substantiate
`such structures in cyclic peptides [52,53].
`In
`addition the configuration in the cyclic structure
`is restricted by the structural bonds and any
`possible b-turn is very specific and can not be
`altered unless the cyclic structure itself
`is de-
`structed.
`The explanatory remarks mentioned above could
`be summarized in the following points:
`
`– No single CD spectra could be attributed to the
`conformation designate b-turn [51].
`– Possible b-turn configuration in cyclic peptides
`such as surfactin need X-ray diffraction and NMR
`for corroboration [52,53].
`– Any possible b-turn in a cyclic peptide is definite
`and will not transform unless the cyclic structure
`is wrecked.
`
`the surfactin
`Since the cyclic structure of
`molecule was maintained all the time under the
`experimental conditions, then the observed gradual
`alterations in the CD spectra of surfactin that are
`described later on in this paper are related to the
`aggregational behaviour of surfactin and could not
`be associated with the possible b-turn configura-
`tion attributed to the cyclic structure of the sur-
`factin molecule.
`
`© 1998 European Peptide Society and John Wiley & Sons, Ltd.
`
`J. Peptide Sci. 4: 449–458 (1998)
`
`PETITIONERS
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`EXHIBIT NO. 1017 Page 3 of 10
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`452
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`OSMAN ET AL.
`
`Effect of the Nonionic Surfactant (C12E7) on the
`Assembly, Conformation and Micellation of Surfactin
`Molecules
`
`Reports by other authors suggest that the addition
`of synthetic surfactants to a peptide solution induce
`conformational transitions in their secondary struc-
`ture [34,54,55]. However, little is known about the
`synthetic surfactants as enhancers or stabilisers of
`defined micellar conformation. Since nonionic sur-
`factants are used in a wide range of applications, we
`have therefore tested the effect of the nonionic sur-
`factant heptaethylene glycol (C12E7) as a model for
`this group of surfactants on the secondary struc-
`ture of surfactin micelles. Contrary to our anticipa-
`tion,
`the micellar aggregation number became
`much higher than expected which indicated an en-
`hancement of
`the assembly of surfactin (Sf)
`molecules in b-sheets and an increase in micelle
`formation. The values of micellar weight-average
`and aggregation number were calculated by the
`extrapolation of the Zimm plots obtained from the
`static light scattering measurements [23]. Figure 1
`shows the changes in micellar weight and the aggre-
`gation number at different Sf:C12E7 ratios.
`As is shown the aggregation number was in-
`creased at the Sf:C12E7 molar ratio of 25:75 and the
`aggregation number raised to 144, indicating en-
`hancement of micellation. Taking the u values for
`the molar elipticity shown in Table 1 into consider-
`ation it becomes clear that the C12E7 enhanced the
`formation of micelles by promoting the assembly of
`surfactin molecules in b-sheets even at very low
`surfactin concentrations of 0.75 mM.
`Table 1 shows the increase of the molar elipticity
`value u at the single minima of 218 obtained from
`the CD spectra measurement in the presence and
`absence of C12E7 at different temperatures.
`Surprisingly, the synthetic surfactant C12E7 did
`not induce any conformational transitions but to
`the contrary, it enhanced the formation of b-sheets.
`Table 1 shows that at 25°C the u value is 1.62(cid:29)
`10(cid:28) 7 deg cm2 dmol(cid:28) 1 in the absence of C12E7 for
`the 3 mM surfactin solution, but this value in-
`creased to 1.71(cid:29)10 (cid:28) 7 deg cm2 dmol(cid:28) 1 at the lower
`surfactin concentration (1 mM) when C12E7 was
`added in a 50:50 Sf:C12E7 ratio The u value in-
`to 1.85(cid:29)10(cid:28) 7 deg cm2
`creased even further
`dmol(cid:28) 1, when surfactin concentration was reduced
`to 0 75 mM and C12E7 was added in a 25:75 Sf:
`C12E7 ratio. This indicates that the b-sheets are
`formed even at very low surfactin concentration due
`to the enhancement caused by the addition of the
`
`synthetic surfactant C12E7. These observations may
`be explained by possible intercalation of the non-
`ionic surfactant between surfactin molecules in the
`micelle, where they possibly function as a template
`that directs the assembly of surfactin in b-sheet
`micelles and thus enhances the b-sheet formation.
`
`Effect of Temperature on the Conformation and
`Assembly of Surfactin Molecules
`
`We further studied the effects of temperature eleva-
`tion on the piling of surfactin molecules in the
`micelles, the secondary structure and the formation
`of b-sheet-micelles, both in presence and in ab-
`sence of C12E7. Our observations clearly indicated
`that increasing the temperature enhanced the for-
`mation of b-sheets both in the absence and in the
`presence of the synthetic surfactant. It also showed
`an increase in micelles aggregation as well as in
`their stability.
`
`Figure 1 The changes in micellar weight and the aggrega-
`tion number of surfactin at different Sf:C12E7 ratios and
`the enhancement of b-sheet micellation.
`
`© 1998 European Peptide Society and John Wiley & Sons, Ltd.
`
`J. Peptide Sci. 4: 449–458 (1998)
`
`PETITIONERS
`
`EXHIBIT NO. 1017 Page 4 of 10
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`

`

`Table 1 Molar Elipticity Changes of Surfactin
`
`TUNING MICELLES OF A PEPTIDE BIOSURFACTANT
`
`453
`
`Substance
`
`Surfactin (Sf)
`
`Sf:C12E7 (molar
`ratio 50:50)
`
`Sf:C12E7 (molar
`ratio 25:75)
`
`Concentration of surfactin
`in the solution (mM)
`
`Temperature
`(°C)
`
`Molar ellipticity [u] deg cm2 dmol(cid:28)1
`at 218 nm minima
`
`3
`3
`3
`2
`2
`1
`1
`
`1
`
`1
`0.5
`0.5
`
`0.75
`
`0.75
`0.5
`0.5
`0.25
`0.25
`
`25
`40
`55
`25
`40
`25
`40
`
`25
`
`40
`25
`40
`
`25
`
`40
`25
`40
`25
`40
`
`1.62(cid:29)107
`1.71(cid:29)107
`1.83(cid:29)107
`2.04(cid:29)107
`2.08(cid:29)107
`1.80(cid:29)107
`1.86(cid:29)107
`
`1.71(cid:29)107
`
`1.73(cid:29)107
`1.63(cid:29)107
`1.73(cid:29)107
`
`1.85(cid:29)107
`
`1.99(cid:29)107
`1.32(cid:29)107
`1.32(cid:29)107
`1.63(cid:29)107
`1.19(cid:29)107
`
`From the results shown in Table 1 we estimated
`maximal increase of 13% in the b-sheet formation
`due to temperature raise. The maximum enhance-
`ment caused by heating of the surfactin system was
`reached at surfactin concentration of 2 mM and at
`temperature of 40°C in the absence of C12E7. The
`maximum temperature effect on enhancing b-sheet
`formation in presence of C12E7 was reached when
`surfactin concentration was 0.75mM, the Sf:C12E7
`ratio was 25:75 and the temperature was 40°C.
`This micellation enhancement
`induced by in-
`creased temperature is in contradiction to what is
`known in the case of normal surfactants. In surfac-
`tant systems employing normal synthetic surfac-
`tants, heating of the surfactant in the aqueous
`solution leads to the de-micellation of aggregated
`surfactant molecules and increase of the dissocia-
`tion of surfactant molecules and hence formation of
`larger number of monomers in the solution.
`The enhancement of micellation by increased
`temperature for this peptide biosurfactant indicates
`strongly that the micellation:de-micellation (associ-
`ation:dissociation) of peptide surfactants is most
`likely ruled by different kinetics from those conven-
`tionally comprehended and applied in the case of
`normal surfactants. It also clearly indicates that the
`theoretical approaches traditionally applied to ex-
`
`plain the behaviour of normal surfactant systems
`may have feasible difficulties in explaining phenom-
`ena such as the enhancement of micellation by
`heating the surfactant in aqueous solutions.
`
`Effects of pH on the Induction of Conformational
`Transitions
`
`Variation in pH is an easy tool for changing proper-
`ties, structure and behaviour of surfactant systems.
`We have examined the effects of pH on the confor-
`mation of surfactin in both micellar and non-micel-
`lar solutions, as it is depicted in Figure 2A. The
`monomers were examined in solutions of 1(cid:29)10 (cid:28) 6
`M, which corresponds to 1:10 cmc of surfactin, and
`the micelles in solutions of 2(cid:29)10(cid:28) 5 M, which corre-
`sponds to about twice the cmc value. Below the cmc
`the surfactin monomers have an unordered confor-
`mation in alkaline solutions when pH is 8.5 or
`more. The unordered structure is distinguished by
`a CD spectra having a strong minima at 202 nm
`and a maxima around 190 nm. At neutral pH the
`conformation changed to b-sheets with a single
`minima at 220 nm and a maxima at 194 nm. In
`slightly acidic pH (pH 6) the surfactin monomers
`have an a-helical conformation, with two minima at
`203 and a stronger minimal value at 222 and a
`maximal value at about 190 nm.
`
`© 1998 European Peptide Society and John Wiley & Sons, Ltd.
`
`J. Peptide Sci. 4: 449–458 (1998)
`
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`454
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`OSMAN ET AL.
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`Figure 2 Effect of pH on the conformation of surfactin molecules in an aqueous solution, as determined by CD. A,
`surfactin monomers; and B, surfactin micelles.
`
`However, different transient conformations were
`observed between pH values shown in Figure 2A.
`This indicated a gradual change from one confor-
`mation to another. The minima around 203 nm and
`222 nm in the CD spectra of these transient struc-
`tures
`indicated that
`the
`surfactin molecules
`adapted a mixed conformation of both b-sheets and
`a-helices where the percentages of each form was
`pH dependent. Thus, the change was gradual and a
`sudden shifting from one form to another was never
`observed. This gradual alteration was most likely
`due to the formation of surfactin clusters (dimers,
`trimers, etc.) by the gradual reduction in pH value.
`Above the cmc the pH effect was quite different. At
`an alkaline pH of 9 or more a-helices were formed.
`These were characterized by a CD spectra having
`two minima at 212 nm and 228 nm as well as
`shoulder minima at 206 and a maxima close to 190
`nm. While at lower pH values of less than 9 and as
`low as pH 6 b-sheets were formed. The CD spectra
`had typically a single minima at 220 and a maxima
`about 195–200 nm. This indicated that normally
`the surfactin micelles would have a b-sheet struc-
`ture when pH conditions are similar to those ob-
`tained under the physiological condition. Figure 2B
`shows the CD spectra of surfactin micelles at differ-
`ent pH values.
`Since surfactin has been shown to be most bioac-
`tive at physiological pH values [8,9,21] and has
`
`adopted the b-sheet conformation under the same
`pH values according to the above results, thus it
`may be concluded that the most favourable bioac-
`tivity would be attained when surfactin micelles
`retain the b-sheet structure. However, this confor-
`mation may be affected by the presence of metal
`ions, where the type and concentration of metal
`ions may enhance or dwindle the assembly in b-
`sheet conformation.
`It may be worth while mentioning that at pH
`values above 9 the cyclic lactone ring of surfactin
`may be cleaved to form linear surfactin in the solu-
`tions stored for relatively longer periods of time
`(data not published), and therefore observations
`above pH 9 may be related to linear surfactin rather
`than to cyclic surfactin [47].
`
`Effect of Ca2(cid:27) on Inducing Conformational
`Transitions in Surfactin
`
`Effects of Ca2(cid:27) and other ions on the restoration or
`repression of the biological activity of surfactin have
`been reported [17,20], and the role of ions in the
`formation of surfactin micelles have also been re-
`ported in the literature [20]. Hosono et.al. [17] ob-
`served an increase in the inhibitory activity of the
`deactivated-surfactin on cAMP phosphodiesterase
`by Ca2(cid:27). Thimon et al. [20] observed that the for-
`mation of surfactin micelles increased the antifun-
`gal activity of
`iturins, but excessive amounts of
`
`© 1998 European Peptide Society and John Wiley & Sons, Ltd.
`
`J. Peptide Sci. 4: 449–458 (1998)
`
`PETITIONERS
`
`EXHIBIT NO. 1017 Page 6 of 10
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`

`TUNING MICELLES OF A PEPTIDE BIOSURFACTANT
`
`455
`
`Figure 3 CD spectra for surfactin monomers and micelles in aqueous solution at different Ca2(cid:27) concentrations and at pH
`of 7.5. A, surfactin monomers; and B, surfactin micelles.
`
`ions decreased the synergism of surfactin
`metal
`interacted with iturins [20]. Yet possible relation-
`ship between ions and the preferred bioconforma-
`tion in surfactin were not examined We have
`investigated the effects of Ca2(cid:27) on the b-sheet con-
`formation of surfactin in both monomeric form (1(cid:29)
`10(cid:28) 6 M solutions) and micellar form (2(cid:29)10 (cid:28) 5 M
`solutions) in aqueous solutions of pH 7.5.
`The transitions induced in the monomers were
`dependent on the concentration of Ca2(cid:27). When the
`Ca2(cid:27) concentration was below 0.3 mM, a-helices
`were formed with two minima at 205 and 225 nm,
`and a maximum around 195 nm. At concentration
`of 0.3 mM the CD showed a strong minimal value at
`198 nm indicating an unordered structure. Such
`strong minima also means that a transition from
`one conformation to another is about to take place.
`At concentrations above 0.3 mM the CD spectra of
`monomeric solution showed a b-sheet conformation
`with a minimal value at 220 nm and a maximal
`value around 190 nm. Figure 3A shows the CD
`spectra for surfactin monomers at the different
`Ca2(cid:27) concentration.
`The transitions observed are likely due to the
`binding of surfactin to Ca2(cid:27) and formation of sur-
`factin clusters.
`The transitions induced by Ca2(cid:27) in surfactin mi-
`celles were totally different from those we observed
`in the monomers. At concentrations below 0.5 mM
`
`a-helical structures were observed, with character-
`istic CD spectra having two minima at 202–206 nm
`and at 222 nm, and a maximal value at 190 nm. At
`a concentration of 0.5 mM Ca2(cid:27) the CD spectra
`showed a strong minimum at 202 nm, indicating an
`unordered structure. This minimum also evidenced
`the start of transition from a-helix to b-sheet struc-
`ture. At concentrations above 0.5 mM, the Ca2 (cid:27)
`ions stabilised the formation of b-sheets with char-
`acteristic CD spectra having a single minimum at
`220 nm and a maximum at 195 nm. Figure 3B
`shows the CD spectra of surfactin micelles at differ-
`ent Ca2(cid:27) concentrations.
`These observations show that the Ca2(cid:27) induces
`a-helical structure both in monomers and in mi-
`celles of surfactin, but at critical concentration
`above 0.3 mM for monomers and above 0.5 mM for
`micelles, Ca2(cid:27) causes a transition to b-sheet
`structure.
`The above observations indicated that calcium
`ions not only affect the conformation of surfactin
`both in monomeric and micellar form, but they also
`induce concentration-dependent transitions. It is
`most likely, that Ca2(cid:27) ions in aqueous solutions of
`surfactin function as templates that direct the clus-
`tering, micellation, aggregation or assembly as well
`as conformation of surfactin molecules, and this
`on the Ca2 (cid:27)
`function is
`quite dependent
`concentration.
`
`© 1998 European Peptide Society and John Wiley & Sons, Ltd.
`
`J. Peptide Sci. 4: 449–458 (1998)
`
`PETITIONERS
`
`EXHIBIT NO. 1017 Page 7 of 10
`
`

`

`456
`
`OSMAN ET AL.
`
`A comparison of our present results with those of
`other authors [17,20] suggests that the preferred
`bioactive conformation of surfactin is likely to be
`the b-sheets. This is because surfactin micelles
`retain the b-sheet structure when calcium is added
`in sufficient amounts to reach concentration ratios
`similar to those reported [17] and because the
`bioactivity also was retained when the overall re-
`ported experimental conditions [17,20] were also
`comparable with our forced conditions.
`To conclude this discussion, it should be noted
`that since b-sheet enhancement reached a maxi-
`mum at 40°C, the physiological temperature of 37.5
`would be perfect for the induction of bioactively
`conformed surfactin. And because optimum pH val-
`ues for b-sheet formation is close to the physiologi-
`cal one (pH 7) and due to the possibility of directing,
`stabilising and enhancing the b-sheet formation by
`other molecule such as Ca2(cid:27) or C12E7. Thus, the
`surfactin micelles could be tuned to maintain the
`most biologically active conformation by inducing
`and stabilising the b-sheet as a secondary struc-
`ture. Since pH and temperature under the physio-
`logical conditions are fairly stable, then micelles,
`microspheres or microemulsions of surfactin or
`other peptide biosurfactants could be probably de-
`signed and preconformed in a bioactive form in
`advance, using Ca2(cid:27) or other assembly directing
`molecules. Otherwise the tuning for non-physiologi-
`cal purposes could be controlled by pH manipula-
`tion, exploitation of directing molecules such as
`Ca2(cid:27), temperature or a combination of these.
`According to the above presented results, discus-
`sion and remarks both Ca2(cid:27) and C12E7 are most
`likely functioning as assembly templates that direct
`the micellation, surfactin micelles could be as-
`sumed as biologically active protein, structured by
`template-aided
`aggregation
`or
`template-aided
`molecular assembly. And consequently we may sug-
`gest the use of ions or other suitable templates to
`direct the micellation, preparation of micro-emul-
`sions, or micro-sphere devices. Such a template-as-
`sembled micelles (TAM) approach could be used to
`direct the formation of not only micelles but also
`microemulsions, micro-spheres (of specific size, di-
`mensions and function) and other molecular
`devices of potential industrial application such as
`drug delivery systems (DDS), drug targeting, micel-
`lar catalysis and in many industrial applications. In
`such process metal
`ions could be used as ionic
`templates in an ion-aided micellation (IAM), but
`other molecules specially designed as templates to
`aid formation of micelles or micro emulsions could
`
`be used in a template-aided micellation (TAM)
`approach.
`
`CONCLUSIONS
`
`It has been made clear that the peptide biosurfac-
`tant surfactin does not follow the normal kinetics of
`surfactant aggregation because the micellation was
`enhanced by increasing the temperature. Another
`deviation from the normal kinetics was the en-
`hancement of aggregation by the synthetic surfac-
`tants. Comparing
`our
`results
`regarding
`the
`conformation of surfactin with the conditions used
`by other authors to test the bioactivity of surfactin it
`became clear that the b-sheet micelles were most
`likely the best bioactive form of surfactin. The b-
`sheet conformation could be easily retained by ma-
`nipulation of extrinsic environmental factors such
`as temperature, pH or Ca2(cid:27). There were also strong
`indications that substances such as Ca2(cid:27) and
`C12E7 direct the micellation and function as assem-
`bly templates for the aggregation process. These
`indications were confirmed by the enhancing b-
`sheet micellation as well as micelle stabilization
`using such substances. Due to these observations
`the tuning of micelles either by environmental ma-
`nipulation or by template-aide aggregation (TAM)
`was suggested. This approach could make possible
`the preparation of micelles, microspheres or microe-
`mulsions with specific dimension, function, stabil-
`ity and bioactivity.
`
`REFERENCES
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`© 1998 European Peptide Society and John Wiley & Sons, Ltd.
`
`J. Peptide Sci. 4: 449–458 (1998)
`
`PETITIONERS
`
`EXHIBIT NO. 1017 Page 8 of 10
`
`

`

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