Langmuir l996,11,4341-4346
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Thermodynamic Analysis of the Surface Activity Exhibited by a Largely Hydrophobic Peptide Gerhard Schwarz* and Susanne E. Taylor Department of Biophysical Chemistry, Biocenter of the University of Basel, Klingelbergstrasse 70, CH 4056 Basel, Switzerland Received May 19, 1995. In Final Form: August 14, 1995@ The putative fusion peptide (23amino acid residues)of the HIV-1virus is markedly hydrophobic,implying poor solubility in water. On the other hand, it gives rise to a pronounced degree of surface activity. We present here a seriesof pertinent surfacepressure data at an airlwaterinterface. Applying a novel evaluation procedure based on thermodynamic principles, the partitioning equilibriumbetween the monolayer phase and the bulk aqueous moiety could be determined in spite of the rather small concentrationin the latter surroundings. The results are quantitatively discussed in terms of the chemical potential and related thermodynamic properties of the interfacial state. It is in particular shown that beyond a surfacepressure of about 15 mN1m the monolayer-associated peptide undergoes a transition to a structure having a smaller effective cross-section(apparently owing to a change of orientation). We propose that our present approach may be extended to peptiddipid mixtures and should as well be more generally applicable to other poorly soluble surfactants. Introduction Significant functions of biological cell membranes, e.g. pore formation, signal transduction, or fusion processes, may be controlled by specific small peptides (comprising a sequence of some 10-40 amino acid residues). This implies that such molecules have a certain extent of hydrophobicity to ensure a sufficient degree of association with the lipid bilayer matrix of a membrane. In order to understand the physical chemistry of molecular mechanisms involving membrane active peptides, the thermodynamic and kinetic properties of their interactions with lipids is an issue of fundamental interest. If those molecules are still reasonably soluble in water, pertinent data can be derived from partitioning experiments carried out by means of titrating an aqueous peptide solution with a liposome suspension. The appropriate procedures and theoretical analyses have been demonstrated with various pore-forming peptides, particularly the bee venom factor melittinl and the wasp venom constituent mastoparam2 This subject has been generally reviewed elsewhere.3 The same approach is technically not feasible in the case of highly hydrophobic peptides that are only poorly soluble in water. Such a situation holds true for the putative hsion peptide of the human immunodeficiency virus type 1 (HIV-11, comprising the sequence Ala-ValGly-Ile-Gly-Ala-Leu-Phe-Leu-Gly-Phe-Leu-Gly-Ala-~aGly-Ser-Thr-Met-Gly-Ala-A~-g-Ser.~ I t represents the 23 amino acid residues at the N-terminus of the viral gp41 transmembrane protein that is presumed to play a decisive role in the fusion of the virus with its target cell.5 The peptide has been shown to produce a pronounced surface pressure at a clean aidwater interface and also to incorporate in a phospholipid monolayer.6 Accordingly a thorough quantitative analysis of these features was * Author to whom correspondence should be addressed. Tel.: 1-41-61-2672200. Fax: +41-61-2672189. Abstract published inAdvanceACSAbstructs, October 15,1995. (1) Schwarz, G.; Beschiaschvili, G. Biochim. Biophys.Acta 1989,970, @
82. (2) Schwarz, G.; Blochmann, U. FEBS Lett. 1993,318,172. (3) Schwarz, G. Biophys. Chem. 1996,in press. (4) Gordon,L. M.; Curtain, C. C.; Zhong, Y. C.; Kirkpatrick, A.; Mobley, P. W.; Waring, A. J. Biochim. Biophys. Acta 1982,1139,257. (5)Larsen, C.; Ellens, H.; Bentz, J. InAdvances in MembraneFluidity;
Aloia, R. C., Curtain, C. C., Gordon, L. M., Eds.; Wiley-Lias. Inc., 1992; Vol. 6, p 1133.
expected to give important insight into the relevant molecular characteristics of the membrane-active substance under consideration. In this article we are concerned with a large-scale series of surface pressure data obtained for a number ofdifferent amounts of the pure peptide without added lipid. The original results are processed in an apparently novel way based on thermodynamic principles. This way we can determine the actual surface concentration in the monolayer as well as the remaining concentration in the aqueous subphase. In other words, the partitioning equilibrium of the surfactant between the airlwater interface and the ordinary hydrated state will be accessible. Then the chemical potential and related thermodynamic properties experienced in the interfacial monolayer may be discussed in quantitative terms. We expect our approach to be rather generally applicable in order to analyze analogously other poorly soluble surfactants. With the given hydrophobic peptide it is shown that increasing the surface pressure induces a structural transition toward a state with smaller area requirements. The present approach is expected to be capable offurther development for the more general case of peptideAipid films. Pertinent studies are in progress. Material and Methods Chemicals. The aqueous medium was a McIlvain buffer7consisting of 20.6 mM Na2HP04and 1.9 mM citric acid monohydrate, resulting in a pH of 7.5 with an ionic strength of 60 mM. Water used for buffers and cleaning was doubly ion-exchanged and quartz glass distilled. DMSO (dimethyl sulfoxide, 98%) as well as the buffer components (both commercial grades of highest purity) were purchased from FLUKA. Peptide. The 23-residue N-terminal sequence of gp41 was synthesized on an Applied Biosystems 43QApeptide synthesizer, using the Merrifield solid-phase technique in which a-amino acids were protected by base-labile Fmoc groups. The complete peptide was cleaved from the resin using a mixture containing TFA (trifluoroacetic acid), water, thioanisole, 1,2-ethanedithiol,and phenol [10:0.5: (6) Rafalski, M.; Lear, J. D.; De Grado, W. F. Biochemistry 1990,29, 7917. >(7) McIlvaine, T.C.J.Biol. Chem. 1921,49, 183.
Q~43-~463/95/2411-4341$Q9.0QlQ 0 1995 American Chemical Society
Schwarz and Taylor
4342 Langmuir, Vol. 11, No. 11, 1995 0.5:0.25:0.75 (vlvlvlvlv)~,yielding the peptide amide. The peptide was precipitated with ether, lyophilized, resolved with 10% formic acid, and then passed twice over a Sephadex G-50 column (Pharmacia, Uppsala, Sweden) which had been equilibrated with 10%formic acid at 4 "C. Eventually the crude peptide was purified by semipreparative HPLC using a Brownlee Aquapore Cg reversed phase column (Applied Biosystems, San Jose CA, USA) with a gradient of acetonitrile to isopropyl alcohol in the presence of 0.1% TFA. The purity and identity of the preparation ('98%) were confirmed by HPLC on a Cg analytical column, by sequence analysis using automated Edman degradation on an Applied Biosystems 477A protein sequenator, and by elementary analysis. The peptide was kept irrlyophilized form at -18 "C. Peptide stock solutions were prepared by dissolving ca. 1 mg of peptide in 1 mL of DMSO such that the final concentration of DMSO had no measurable effect on surface pressure. The peptide concentration was determined by performing a periodically repeated quantitative amino acid analysis in the presence of a known quantity of norleucine and extrapolating it. The stock solution was stored at -18 "C in a Teflon-sealed glass vial. Monolayer Measurements. Two different types of troughs were used: (1)a round Teflon trough like that of Fromherza with a total area of 360 cm2divided into eight compartments (model RMC 2-T; Mayer Feintechnik, Gottingen, Germany), each with a surface area (A) to volume (V, ratio of 1.8cm-l and (2) comparable conventional Langmuir troughs (made by our workshop) with A I Vfrom 0.4 to 1.5cm-l. All our troughs were surrounded by a closed box to avoid contamination and to keep humidity constant. The surface pressure was measured by the Wilhelmy m e t h ~ dusing , ~ hydrophilic platelets cut from filter paper (Whatman No. 1)l0which were rinsed with water after use and stored under water in a closed Teflon box. Before each measurement the trough was thoroughly cleaned with DMSO, ethanol, and water. The possible statistical error in the measured pressure values is estimated to be on the order of 0.1 mN1m. The aqueous bulk solution was stirred with an appropriate magnetic bar to assure a homogeneous distribution of the solute. Small increments (maximum 5 pL) of the peptide stock solution were spread on the surface with a microsyringe (Hamilton, CH-7402 Bonaduz GR, Switzerland)to obtain the desired amount of total peptide. For each experiment the surface pressure was monitored until equilibrium was reached. This situation is indicated by a slow decrease of pressure at a constant rate owing to evaporation. For pressure-area curves ("isothermal mode" of measuring) the round Teflon trough was used with the monolayers being compressed at a speed of 5 cm2/min by moving a Teflon barrier. The conventional troughs were used for measurements at constant area with no further accessibleair-water interface ("isochorous mode"). All measurements were performed at room temperature (22 f 1 "C) and atmospheric pressure. Evaporation. The evaporation rate of water was determined to be 4 x mL/cm2 min, based on monitoring the weight of a buffer-filled trough with a monolayer of peptide. For isochorous measurements the total volume has been correspondingly adjusted. Corrected values of the surface pressure were calculated according to the observed slope angle of its time course. However, prior to measuring a pressure-area isotherm the surface pressure was set to zero so that the pressure had not to be corrected for evaporation. (8) Fromherz, P. Reu. Sci. Instr. 1976, 46, 1380. (9) Wilhelmy, L. Ann. Phys. Chem. 1863,119, 177. (10)Gaines, G. L. J. Colloid Interface Sci. 1977, 62, 191.
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Figure 1. Amounts (nanomoles)of total peptide, np, versus the interfacial area,A, at fured volume, V, and lateral pressure, n = 15 mN/m ( 0 )and 35 mN/m ( x ), respectively.
Thermodynamic Equilibrium. To be sure that the monolayers were in thermodynamic equilibrium and no exchange of peptide took place through the bulk into the arising free airlwater interface when moving the barriers, our data were compared with isochorous measurements made without barriers in conventional troughs having different surface areas. The results turned out to be in good agreement. Also, direct measurements of possible lateral pressures in such free interfaces showed no effect. In addition we did measurements with different accessible Teflon surfaces in order to find out whether the peptide binds to the Teflon wall. Measurable Teflon effects could, however, not be detected. Processing of Data. In an individual experiment the surface pressure, n,had been measured with a given total amount, np, of peptide (on the order of 1 nmol) for a gradually decreasing trough area, A. We have recorded a series of n versus A isotherms applying to a number of sufficiently different values of np. Then pairs of np and A taken from all the available isotherms at the same surface pressure were collected. Because of mass conservation, these quantities must be subject to the relation
where r is the relevant monolayer surface concentration and c b stands for the aqueous bulk concentration in the subphase at a total volume V. Under equilibrium conditions r as well as c b is invariant at fmed n owing to thermodynamic principles (see the Appendix). Accordingly, a plot of npversus A must be linear (provided Vand further possiblydecisivephysical parameters, particularly temperature and pH, are held constant). The slope of this straight line must be equal to r, whereas c b can be determined from the intercept on the ordinate. Our data have indeed verified the proposed linear relationships with remarkably little uncertainty. This is demonstrated by two examples in Figure 1. Results The course of n versus A was registered for seven total peptide amounts, namely np= 0.47 (only up to 25 mN/m), 0.79, 1.10, 1.54, 1.85, 2.17, 2.32 nmol. We then plotted these npvalues versus the appropriate interfacial areas A for fixed lateral pressures n = 1, 3, 5, 7, 10 mNIm and
Surface Activity of a Largely Hydrophobic Peptide
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ap [nm2/molecule] 30
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Figure 3. Area per peptide molecule, a,,, in the monolayer versus surface pressure as evaluated from the data of Figure 2. The solid curve has been calculated with our model (r,,,r, being indicated by the dashed borderlines). In the inset
1
l n n A t q q A < m n l o n A n w n a n nnn+;finn nf+hn;Jon1 n-hnlio-1 0nnfn-Sluuglbuuulalauu b~uuu ~ ~ S C . U L U I L UVL u i ~ LiUUUI U-LLUIAUU~ .-'___________------tion are shown (dark zone, hydrophilic part; light zone, I
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UwAAAwA
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Figure 2. Surface pressure, x, as it functionally depends on
the actual surface concentration, r (determinedby the slope of a linear plot according to the examples in Figure 1). The lower dashed line follows x i d =RZT. The solid fitting curve has been calculated by means of the theoretical model described in the Discussion section (the vertical dashed line indicates r, = 49 pmol/cm2).
subsequently in steps of 1 mN/m up to 41 mN/m (see the examples in Figure 1). By linear regression analysis (with excellent correlation coefficients around 0.9999) the surface concentration r (slope)and the bulk concentration c b (ordinate intercept divided by the given volume) could be determined according to the relation presented in eq 1. Because of the quality of the obtained straight lines, the inherent statistical errors of r and c b are rather limited. At low and high lateral pressure (
