Orientational Distributions of the Di-r-helical SyntheticPeptide ZnPPIX-BBC16 in Langmuir Monolayers by X-ray

Reflectivity and Polarized Epifluorescence

A. Tronin,*,† J. Strzalka,† X. Chen,‡,§ P. L. Dutton,‡ B. M. Ocko,| and J. K. Blasie†

Chemistry Department, University of Pennsylvania, Philadelphia, Pennsylvania 19104,Department of Biochemistry and Biophysics, University of Pennsylvania,

Philadelphia, Pennsylvania 19104, and Department of Physics,Brookhaven National Laboratory, Upton, New York 11973

Received October 23, 2000. In Final Form: February 21, 2001

The vectorial orientation of the peptide ZnPPIX-BBC16 in a Langmuir monolayer was studied by meansofX-rayreflectivityandpolarizedepifluorescence.ZnPPIX-BBC16consistsof two R-helical31-mersdimerizedvia a disulfide bridge between two N-terminal cysteines (R-S-S-R), so that two bis-his metalloporphyrinbinding sites between positions 10,10′ and 24,24′ are formed. Zn(II) protoporphyrin IX was bound to theposition 24. To enhance stability at the interface, a palmitoyl (C16) chain was bonded to each N-terminalcysteine. X-ray reflectivity measurements make it possible to infer the orientation of the R-helices bydetermining the electron density profile of the monolayer. Polarized epifluorescence provides the orientationdistribution of the porphyrin with respect to the monolayer plane. Both X-ray and fluorescence measurementsshow that at low surface pressure (π ) 5 mN/m), the peptide R-helices lie in the plane of the water surfacewith a narrow orientational distribution of the porphyrin. At high pressures (π > 30 mN/m), the peptideR-helices are perpendicular to the water surface and the distribution is wider. At intermediate pressuresthe peptide R-helices in the monolayer undergo a transition from the parallel to perpendicular orientationwith almost a complete loss of the orientational order of the porphyrin.

1. IntroductionMonolayer films of oriented natural or synthetic proteins

containing metalloporphyrin, a major effector of biologicaloxidation-reduction reactions, are very promising forfundamental biophysical studies and biotechnology ap-plications. One of the well-established techniques forfabricating ordered monolayers on solid supports is theLangmuir-Blodgett (LB) method. The structure of a LBfilm is determined to a substantial degree by that of theprecursor Langmuir monolayer formed at the air-waterinterface. In the case of protein Langmuir films, theorientation of the molecules in the monolayer depends toa great extent on external parameters such as surfacepressure. It has been shown that reorientation due tochanges in surface pressure occurs in monolayers com-posed of very different proteins, such as immunoglobulinG,1 cytochrome p450ssc,2 photosynthetic reaction centers,3and the synthetic peptide BBC16.4 Thus, the LB techniquemakes it possible to control molecular orientation viasurface pressure, which can be very important for makingfunctional films. Knowing the orientation of the moleculesin the precursor Langmuir monolayer at the air-waterinterface is therefore very important for creating LB filmswith desired structure. A very powerful technique for

obtaining structural information about a monolayer isX-ray reflectivity (XR), which can determine the electrondensity profile of a monolayer.5 Another promising tech-nique for studying molecular orientation is the recentlyintroduced polarized epifluorescence (PEF) technique.6In this technique, the orientation of the fluorophore (Zn-porphyrin in this case) can be determined by measuringthe polarization of the fluorescence excited by an electricfield directed normal to and along the film surface. Themain advantage of fluorescence measurements over otherlinear optical techniques is that fluorescence, a “two-photon” process, makes it possible to determine not onlythe mean orientation angle of the porphyrin with respectto the monolayer plane but also the width of the orien-tational distribution.6-8 This feature is of particularimportance since it gives a measure for the degree of theorientational order of the monolayer. Both XR and PEFcan be applied to the study of monolayers at the air-water interface and provide complementary informationon the orientation of the peptide itself and the functionalgroup, respectively. Recently we have studied the Lang-muir monolayers of apoBBC16 (i.e., BBC16 withoutmetalloporphyrin bound).4 We showed that the peptideorientation changes with surface pressure. At low pressure(π < 22 mN/m) the peptide is oriented with its diheliceslying in the plane of the monolayer, whereas at highpressure (π > 30 mN/m) the peptide’s dihelices areperpendicular to the surface. We were not able to inferany knowledge about the orientational order, since theabsence of the suitable fluorophore did not permit the

* To whom correspondence may be addressed. E-mail:tronin@sg1.chem.upenn.edu.

† Chemistry Department, University of Pennsylvania.‡ Department of Biochemistry and Biophysics, University of

Pennsylvania.§ Present address: Department of Chemistry and Chemical

Biology, Harvard University, 12 Oxford St., #292, Cambridge, MA02138.

| Department of Physics, Brookhaven National Laboratory.(1) Tronin, A.; Dubrovsky, T.; Nicolini, C. Langmuir 1995, 11, 385.(2) Guryev O.; Dubrovsky T.; Chernogolov A.; et al. Langmuir 1997,

13, 299.(3) Facci P.; Erokhin V.; Paddeu S.; et al. Langmuir 1998, 14, 193.(4) Strzalka, J.; Chen, X.; Moser, C.; Dutton, P. L.; Ocko, B. M.; Blasie,

J. K. Langmuir, in press.

(5) Als-Nielsen, J. In Handbook on Synchrotron Radiation; Brown,G. S., Moncton, D. E., Eds.; Elsevier Science Publishers: Amsterdam,1991; Vol. 3, p 471.

(6) Tronin, A.; Strzalka, J.; Chen, X.; Dutton, P. L.; Blasie, J. K.Langmuir 2000, 16, 9878.

(7) Edmiston, P. L.; Lee, Cheng, S.-S.; Saavedra, S. S. J. Am. Chem.Soc. 1997, 119, 560.

(8) Bos, M. A. and Kleijn, M. Biophys. J. 1995, 68, 2573.

3061Langmuir 2001, 17, 3061-3066

10.1021/la001489c CCC: $20.00 © 2001 American Chemical SocietyPublished on Web 04/18/2001

fluorescence measurements. In the present paper we studythe monolayers of the porphyrin containing or holoformof the same peptide, ZnPPIX-BBC16. The structure ofthese two forms is very similar, so we expect similar resultsfor the ZnPPIX-BBC16. Presence of the Zn-porphyrin inthe peptide structure allows us to apply PEF.

The synthetic peptide was designed de novo as a modelfor intramolecular electron transfer. The synthetic unitis an R-helical 31-mer with a palmitoyl (C16) chain bondedto the N-terminal cysteine to increase the peptide’samphiphilicity. The units dimerize via a disulfide bridgebetween two cysteines (R-S-S-R) to form two bis-hismetalloporphyrin binding sites between sequence posi-tions 10,10′ and 24,24′. The schematic peptide structureis shown in Figure 1. We define the peptide frame by thethree axes ê, ψ, and ú. The approximate dimensions of thepeptide group, without the hydrocarbon chains, are 50,20, and 10 Å, respectively. We will call the (ê, ψ) plane thepeptide plane, referring to the largest, smallest, andmedium dimensions as “length”, “thickness”, and “width”of the molecule. Pronounced differences in the length andthickness make it possible to infer the molecular orienta-tion from the measurements of the monolayer electrondensity profile.4 The porphyrin plane is perpendicular tothe ψ-axis. The metalloporphyrins shown occupy bothbinding sites. In solution, however, the metalloporphyrinaffinity to the first site is low possibly due to interferencefrom the palmitoyl chain, and it is possible to bindporphyrin in the 24,24′ position only. However, uponsubsequent spreading in a Langmuir monolayer, thisinterference should be removed resulting in the metal-

loporphyrin’s redistribution to occupy both the 10,10′ and24,24′ binding sites.

2. Materials and Methods

BBC16 was synthesized according to ref 9. ZnIIPPIX waspurchased from Porphyrin Products, Inc. To bind ZnIIPPIX toBBC16, the equivalent of 1 porphyrin per 1 dihelix was titratedinto a 0.1 mM BBC16 solution in 1 mM phosphate buffer with10 mM NaCl, pH 8, from a 2.7 mM solution of ZnIIPPIX in DMSOin accordance with ref 10. The concentration of the peptidesolution was determined spectroscopically by measuring theabsorption at λ ) 280 nm.10,11 The titration of ZnIIPPIX wasmonitored spectroscopically by observing the red shift of the Soretabsorption band from 404 nm for free ZnIIPPIX to 427 nm, whichcorresponds to the metalloporphyrin in bis-his coordination. Theresulting solution was used for spreading the Langmuir mono-layer. To prepare the monolayer of ZnPPIX-BBC16, a techniquedescribed in ref 4 was used with the exception that in the presentstudy we used only pure peptide monolayers without any additionof a phospholipid. X-ray reflectivity measurements were per-formed at beamline X22B of the National Synchrotron LightSource using a previously described Liquid Surface spectrometer4

with a small, custom-built Langmuir trough (Ames Lab, Ames,IA) mounted on the sample stage. XR data were analyzed usingbox refinement (BR), an iterative, model-independent procedurefor recovering the electron density profile, F(Z), of a monolayer

(9) Gibney, B. R.; Rabanal, F.; Skalicky, J. J.; Wand, A. J.; Dutton,P. L. J. Am. Chem. Soc. 1999, 121, 4952.

(10) Sharp, R. E.; Diers, J. R.; Bocian, D. F.; Dutton, P. L. J. Am.Chem. Soc. 1998, 120, 7103.

(11) Robertson, D. E.; Farid, R. S.; Moser, C. C.; Urbauer, J. L.; etal. Nature 1994, 368, 425.

Figure 1. Schematic structure of ZnPPIX-BBC16: (a, b, c) mutually perpendicular projections; (d) isometric projection. ê and ψaxes lie in the plane of the axes of the two R-helices; ê is parallel and ψ is perpendicular to the axes of the two R-helices. The porphyrinplane is perpendicular to the ψ axis.

3062 Langmuir, Vol. 17, No. 10, 2001 Tronin et al.

from the data.12,13 For the details of the BR application to theLangmuir monolayer case see refs 4 and 14.

The polarized epifluorescence measurements were performedwith a custom-built fluorimeter, described in detail elsewhere.6The design makes it possible to acquire fluorescence intensitydata directly from the air-water interface. The Langmuir troughused in epifluorescence measurements was a custom trough fromRiegler & Kirstein GmbH. The wavelength of the excitation beamwas λ ) 514 nm. The fluorescence was observed trough a 570 nmcutoff filter (Melles-Griot). To acquire the background signal forthe fluorescence measurements, monolayers of apoBBC16 oth-erwise prepared identically were used. Acquisition time for eachfluorescence intensity measurement was 5 s. Intensities wererepeatedly acquired in the order Isx, Ipx, Ipy, Isy, Isy, Ipy, Ipx, Isx, etc.(indices p, s indicate the polarization of the excitation field andx, y indicate the polarization of the emitted field), to get sufficientstatistics and to eliminate the influence of porphyrin photo-bleaching.7 The overall errors of the measured intensities dueto the noise and photobleaching were less than 1%.

3. Results and Discussion

3.1. Compression Isotherms. Figure 2, an isothermcollected from a Langmuir monolayer of the ZnPPIX-BBC16, shows how the surface pressure (π) varies as afunction of the area of the monolayer. Compression andexpansion show pronounced hysteresis. There are at leastthree regions with different compressibility that can bedistinguished in the compression curve. The first one,denoted as “a” on the figure, begins with the surfacepressure onset at about 400 Å2 per R-helix and extendsto π ≈ 22 mN/m. This region is characterized by lowcompressibility. The second one (“b”), with much highercompressibility, begins at the end of region a and ends atπ ≈ 30 mN/m, where the curve exhibits the sharp kinkand the compressibility becomes low again. The thirdregion (“c”) begins at this kink. The value for the limitingarea per R-helix for the third region is 100 Å2, which isabout the smallest cross section of the R-helix.

3.2. X-ray Reflectivity. Pronounced variations of themonolayer compressibility suggest some structuralchanges. To get insight in the peptide orientation, we usedX-ray reflectivity measurements in each of these threeregions. Figure 3 shows X-ray reflectivity data, normalizedby the Fresnel reflectivity for an infinitely sharp water-helium interface, taken at five surface pressure values,namely, 5 mN/m (region a), 22 and 27 mN/m (region b),and 30 and 34 mN/m (region c). Experimental data areshown by markers, calculated results from box refinement

(see below) are shown by the continuous curves. One cansee systematic changes in the reflectivity with the surfacepressure. The curves for the regions a and b have onebroad maximum over the range of momentum transfershown, whereas those for the region c have several maximamuch narrower in width over the same range. Throughoutregions a-c, the overall normalized X-ray reflectivity shiftstoward lower values of momentum transfer and thenarrower maxima become more pronounced as π increasessuggesting that the monolayer thickness increases. Thethickness can be determined directly by calculating theautocorrelation of the gradient of the monolayer electrondensity profile. The range in which the autocorrelationfunction has nonzero values yields the maximum extentof the monolayer structure in the direction perpendicularto the surface (Z-direction). One of the main advantagesof the autocorrelation function is that it is computeddirectly from the experimental normalized reflectivity datawithout imposing any a priori conditions. The results ofthe computation are shown in Figure 4. The most evidentdifference is between the curves corresponding to regionsa and c. The autocorrelation functions from the low-πregion a decay to zero by Z ) 20 Å while those from thehigh-π region c extend to about 60 Å and include a secondminimum in the vicinity of 55 Å that is absent in the

(12) Makowski, L. J. Appl. Crystallogr. 1981, 14, 160.(13) Stroud, R. M.; Agard, D. A. Biophys. J. 1979, 25, 495.(14) Zheng, S.; Strzalka, J.; Ma, C.; Opella, S. J.; Ocko, B. M.; Blasie,

J. K. Biophys. J., in press.

Figure 2. Compression-expansion isotherm of the monolayerof ZnPPIX-BBC16. See text for details.

Figure 3. Modulus of the X-ray reflectivity divided by theFresnel reflectivity of pure water vs surface pressure of theLangmuir monolayer of ZnPPIXBBC16: diamonds, π ) 5mN/m; triangles, π ) 22 mN/m; circles, π ) 27 mN/m; crosses, π) 30 mN/m; squares, π ) 34 mN/m. Experimental data areshown by markers; lines show the calculation for the electrondensity profiles determined via the box refinement procedure.

Figure 4. Autocorrelation function for the gradient of theelectron density profile of the Langmuir monolayer of ZnPPIX-BBC16 vs surface pressure.

Peptide Orientation in a Langmuir Monolayer Langmuir, Vol. 17, No. 10, 2001 3063

low-π functions. The curve from region b at π ) 27 mN/mhas a weak second minimum and may represent anintermediate case. Looking at the individual curves fromregions a and b more closely, we see that the location ofthe first minimum in the autocorrelation function varieswith π from Z ) 14 Å at π ) 5mN/m to Z ) 17 Å at π )27 mN/m. In contrast, the location of the second minimumin the functions for region c shows no noticeable depen-dence on surface pressure.

The change in monolayer thickness with surface pres-sure is clearly seen in the electron density profiles obtainedvia box refinement from the XR data (Figure 5). Helium(F = 0) fills the positive half-space while the semi-infinitesubphase (F ) 1) extends to the left. In our calculation wenormalized electron density F(Z) to that of the water, Fw) 0.333 e-/Å2. The region with density F > 1 is the peptidemonolayer. At lower pressures the monolayer thicknesssystematically increases from 10 Å for π ) 5 mN/m to 15Å for π ) 22 mN/m. At higher pressure the thicknessincreases drastically up to 50 Å. Normalized reflectivitycurves, calculated for the resulting electron densityprofiles, are shown in Figure 3 by the continuous curvesand agree well with the experimental data.

3.3. Polarized Epifluorescence. To get more infor-mation on the peptide orientation in the monolayer, weused polarized epifluorescence measurements. PEF andXR measurements were not performed at exactly the samesurface pressures but covered overlapping ranges. PEFmeasurements were done at π ) 5, 22, 32, and 40 mN/m.We define a tilt angle θ as the angle between the normalof the porphyrin ring and the normal of the monolayerplane. Assuming that the tilt angle obeys a Gaussiandistribution

we can apply PEF to measure the mean tilt angle, θm, andthe width of the distribution, σ. The distribution param-eters obtained are given in Table 1. The quality of the fitswas sufficiently good that the discrepancies between themeasured and calculated intensities were less than the

experimental errors. The corresponding calculated Gauss-ian distribution curves are shown in Figure 6. At the lowestpressure the planes of the porphyrins are oriented onaverage perpendicular to the monolayer plane with a highdegree of orientational ordering, i.e., a relatively narrowdistribution width. For π ) 22 mN/m the porphyrinorientation changes dramatically: the mean tilt angledrops to the value of 65° and the distribution becomesvery broad. With further increasing pressure, the tilt angleshifts back toward 90°, and the distribution becomesnarrower. At the maximum surface pressure the averageporphyrin orientation is close to perpendicular again. Itwas shown6 that the mean tilt and the distribution widthas determined by polarized fluorescence are highly inter-related, and the accuracy of the orientation distributiondetermination is given by some range of (θm,σ) valueswhich comply with the fluorescence intensities. Theseregions of allowed distribution parameters are shown inFigure 7. At the lowest surface pressure the mean tilt andwidth are confined to a small region with 75° < θm < 90°and 0° < σ < 18°. For π ) 22 mN/m the region of theallowed θm and σ becomes very broad in both the tilt andwidth with 25° < θm < 90° and 0° < σ < 90°. We note herethat for this intermediate pressure, the determinedGaussian distribution is so broad that the relative numberof porphyrins within the vicinity of the mean tilt angle isonly slightly greater than the numbers at the extremesof the distribution, as shown in Figure 6. It is possiblethat for such broad distributions, a bimodal distributionmight be a better description of the tilt angle distribution,but the number of parameters required for this deter-mination is beyond the current scope of the techniqueemployed. For π ) 32 and 40 mN/m the region is muchmore confined than for π ) 22 mN/m. With an increaseof surface pressure from 32 to 40 mN/m the allowed regionshifts toward higher values of θm and lower values of σ.Although the range of the allowed θm values remainsrather broad, the allowed mean tilt angles for the

Figure 5. Electron density profile of the Langmuir monolayerof ZnPPIX-BBC16 vs surface pressure obtained via a boxrefinement procedure. Zero of the Z axis corresponds to theair-water interface; the positive direction of the axis is towardair. Electron density is normalized to that of the water, so thatthe value of 1 corresponds to the subphase, and the value of 0to the air.

P(θ) )exp(-(θ - θm)2/2σ2)

∫0

πexp(-(θ - θm)2/2σ2) dθ

Table 1. Distribution Parameters for the LangmuirMonolayer of ZnPPIX-BBC16 vs Surface Pressure

surface pressure,mN/m

mean tiltangle, deg

distributionwidth, deg

5 90 822 65 7032 67 5040 74 35

Figure 6. Porphyrin orientation distribution for Langmuirmonolayers of ZnPPIX-BBC16 vs surface pressure as deter-mined from the polarized epifluorescence measurements.

3064 Langmuir, Vol. 17, No. 10, 2001 Tronin et al.

porphyrins must be greater than 50°, since the values ofθm < 50° do not agree with the fluorescence measurementsfor either of the two higher pressures.

4. Discussion

Both X-ray reflectivity and polarized fluorescencemeasurements provide strong evidence that the peptidedihelices lie in the monolayer plane at the lowest pressurestudied (see Figure 8a). The electron density profile showsthat the monolayer thickness is about 10 Å. This valuecoincides with the thickness of only one R-helix while thedihelix would be double that value. The porphyrin ringsare perpendicular to the surface in this case, in agreementwith the fluorescence measurements.

In the range c the monolayer thickness is 50 Å. Thisvaluecoincideswith thepeptide length andcan beachievedby perpendicular orientation of the peptide dihelices(Figure 8b). Note that the thickness is constant in therange of surface pressure 30 < π < 34 mN/m, which allowsus to rule out any kind of the peptide multilayer formation.The perpendicular orientation is supported by the surfacepressure isotherm, because the limiting area in thispressure range coincides with the smallest cross sectionof the R-helix. The porphyrin tilt angle is again about 90°for the perpendicularly oriented peptide, which agreeswith (or at least does not contradict) the results of thefluorescence measurements. The behavior of the mono-layer of the ZnPPIX-BBC16 is similar to that of apoBBC16,studied previously by X-ray reflectivity.4 The latterundergoes a sharper transition from the parallel orienta-tion to the perpendicular one.

In the range a, the monolayer thickness increasesslightly with pressure. This increase could originate fromthe peptide tilting about either the ê or ψ axis (defined inFigure 1). However, any rotation about the ψ-axis doesnot cause changes in the porphyrin tilt angle (Figure 8d).The porphyrin, being perpendicular to the rotation axis,remains in the same plane. In the case of rotation aboutthe ê axis (Figure 8c) the porphyrin tilt angle changes bythe same amount. The increase of the thickness ∆d isrelated to the tilt angle as ∆d ) l cos θm, where l is the

peptide width. For ∆d ) 5 Å and l ) 20 Å, we predict θm) 75°, which agrees reasonably well with the value of θm) 65° obtained from the fluorescence measurements. Thelatter also show that the porphyrin orientation distributionis extremely wide in the region b, which means that thepeptides are almost completely disordered with respectto the rotation around the ê axis.

At π ) 27 mN/m there appears to be a coexistencebetween two peptide orientations. The broad shoulder tothe left of the peak in the electron density profile at thispressure does have a length that corresponds to peptidedihelices oriented perpendicular to the interface. However,the low density of the shoulder, especially as relative tothat for the higher pressures, suggests that the fractionof molecules in the perpendicular orientation is small,and the majority of the monolayer is composed of peptidemolecules whose dihelices remain oriented parallel to theinterface.

To better understand the behavior of the peptide at theair-water interface, it is useful to consider the distributionof the hydrophobic/hydrophilic areas over the surface ofthe peptide molecule. The molecular structure of thedihelical peptide, obtained as a result of solution NMRtechnique15 is shown in Figure 9. The hydrophobic residuesare marked by black. There is a significant amount ofexposed hydrophobic residue, located mostly on one faceof the molecule. Such a distribution should render somesurface activity to the molecule.

The experimental observations and molecular structureallow us to propose the following model for the monolayerstructure. In the surface pressure range a, the peptidedihelices lie in the plane of the surface. The hydrophobicface is directed away from the subphase, providing a highdegree of the orientational order. The compressibility of

(15) Skalicky, J. J.; Gibney, B. R.; Rabanal, F.; Urbauer, R. J. B.; etal. J. Am. Chem. Soc. 1999, 121, 4941.

Figure 7. Uncertainty of the (θm, σ) determination forLangmuir monolayers of ZnPPIX-BBC16: π ) 5 mN/m,diamonds; π ) 22 mN/m, triangles; π ) 32 mN/m, squares; π) 40 mN/m, crosses. For the surface pressure π ) 5 mN/m theregions of possible (θm, σ) values are limited by the shown curveand the line θm ) 90°, for the higher surface pressure the regionsare limited by the respective curves and the lines θm ) 90° andσ ) 90°. Uncertainty is due to errors in the measurement of thefluorescence intensities.

Figure 8. Scheme of the ZnPPIX orientation in the Langmuirmonolayer: (a) low surface pressure, π < 22 mN/m; (b) highsurface pressure, π > 30 mN/m; (c, d) intermediate surfacepressure, 22 < π < 30 mN/m.

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the monolayer being restricted by the molecular interac-tions is low. In the range b the molecules begin to rotatearound the ê axis, the monolayer thickness and com-pressibility increase, and the orientational order of theZn-porphyrins decreases. The hydrophobic faces are nolonger totally exposed to the air; they are forced into thesubphase by the surface pressure. With further pressureincrease the average time during which the hydrophobicfaces contact the water increases (as does the number ofthe molecules with the immersed hydrophobic faces). Oncea sufficient fraction of the molecules become oriented withtheir ψ axis perpendicular to the water surface, thepossibility arises that neighboring immersed moleculescan associate to form dimers in which the hydrophobicfaces are apposed, and the external surface of the dimeris hydrophilic. Such dimers are much less surface active;they are anchored to the interface by the hydrocarbonchains only, and the peptide domains can go into thesubphase without increasing the energy of the system. Atthis point the monolayer structure changes abruptly. Thepeptide dihelices take the perpendicular orientation, andthe monolayer thickness increases to the value of 50 Å.The vectorial orientation is provided by the surface arealimitation in this range, and the orientational orderincreases as the area per molecule decreases. The idea

that the change of the orientation is associated with thedimer formation is supported by the various experimentaldata showing that the dihelical peptides without thehydrocarbon chain forms dimers (“four-helix bundles”) insolution.15

In conclusion we can say that the X-ray reflectivity andpolarized epifluorescence data provide strong evidence ofthe reorientation of the ZnPPIX-BBC16 in the Langmuirmonolayer. At lower surface pressure the peptide’s di-helices lie in the plane of the surface with a high degreeof orientational order. At high pressure they orientperpendicular to the interface but are less well ordered.In the intermediate region the molecules rotate aroundthe dihelical long axis with almost complete loss oforientational order of the porphyrins. It seems plausiblethat the transition from the in-plane to the perpendicularorientation is accompanied by the formation of the dimersof the peptide, i.e., four-helix bundles.

Acknowledgment. This work was supported by NIHGrants GM33525 and GM 41048 and NSF/MRSEC GrantDMR96-32598. Authors are grateful to David Vaknin foruse of his trough and to Elaine DiMasi for extensive helpwith the liquid surface spectrometer.

LA001489C

Figure 9. Distribution of hydrophilic/hydrophobic residues over the surface of BBC16. Hydrophobic residues (Ala, Val, Phe, Pro,Ile, Leu) are shown in black. The views show the molecule at four successive rotations about the ê axis by 90°.

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