Compressibility and Elasticity of Amphiphilic and Acidic β-Sheet

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J. Phys. Chem. B 2011, 115, 50–56

Compressibility and Elasticity of Amphiphilic and Acidic β-Sheet Peptides at the Air-Water Interface Vladimir Vaiser and Hanna Rapaport* AVram and Stella Goldstein-Goren Department of Biotechnology Engineering and the Ilze Katz Institute for Nanoscale Technology, Ben-Gurion UniVersity of the NegeV, P.O. Box 653, Beer-SheVa 84105, Israel ReceiVed: September 6, 2010; ReVised Manuscript ReceiVed: NoVember 8, 2010

Grazing incidence X-ray diffraction measurements were performed on monolayer films of three amphiphilic and acidic β-sheet peptides having the general sequence Pro-Y-(Z-Y)5-Pro, with Y ) Asp or Glu and Z ) Phe or Leu denoted, PFD-5, PLE-5, PLD-5, and the 1:1 molar ratio mixture of PLD-5 and PLE-5. The crystalline domains of these peptides exhibited compressibility and elasticity of the crystalline unit cell indicated by changes in diffraction patterns on compression. Higher compressibility values appeared to be associated with more favorable cross-strand interactions between peptides with the larger side chains, whereas PLD-5, decorated by the smaller side chain amino acids, exhibited the lowest crystalline compressibility. Diffraction patterns provided evidence for a new subunit cell generated by the pleated β-strand motif in an apparently favorable mode of cross-strand intermolecular packing in β-sheets. The study contributes to the understanding of β-sheet flexibility at interfaces with relevance to natural proteins and designed biomaterials composed of β-sheet peptides. Introduction Amphiphilic peptides assuming a specific secondary structure may self-assemble through noncovalent intermolecular interactions into supramolecular architectures. The β-sheet structure is composed of pleated β-strands that are stabilized by interstrand hydrogen bonds and by intermolecular interactions between amino acid side chains. Peptides composed of alternating hydrophilic and hydrophobic amino acids that tend to adopt the β-sheet conformation were shown by various techniques to form two-dimensional ordered monolayers on surfaces.1-3 We utilized grazing incidence X-ray diffraction (GIXD) to characterize peptides that were designed to assemble into β-sheet monolayers at the air-water interface. GIXD provided in situ structural information on lattice spacings and extent of order.1 The diffraction patterns of these two-dimensional crystalline structures display in general two main Bragg peaks, one that is associated with the peptide length and the other with the interstrand spacing that is determined by the hydrogen bonds that link neighboring stands in the β-sheet (Figure 1). Recently, GIXD measurements of the peptide Pro-Glu-(Phe-Glu)5-Pro, PFE5, monolayer that were obtained along compression-expansion Langmuir isotherms indicated compressibility and one-dimensional quasi-reversible elasticity of the β-strands along the peptide long axis direction within the ordered domains.4 The diffraction pattern indicated that the repeat distance of the peptide long axes decreased in length by 37% along film compression, whereas upon expansion the compressed assemblies reverted elastically to their original conformation. Experimental evidence on stress-strains behavior in polypeptide secondary structures may advance our understanding regarding the role of structural flexibility in protein function. Similar aspects have been addressed in studies of single crystals formed at high pressure.5 Compressibility and elasticity are of interest in studies concerning structure versus function and * To whom correspondence should be addressed. Phone: +972-86479043. Fax: +972-8-6477188. E-mail: [email protected].

Figure 1. Schematic representation of the two-dimensional peptide assembly showing the compression and out-of-plane bending of PFD5, Pro-(Glu-Phe)5-Glu-Pro, that on expansion reverts elastically back to the original conformation in correspondence with the applied surface pressure.4

activity of apolipoprotein B (apoB), a large protein that participates in the assembly and secretion of VLDL particles that carry lipids, triacylglycerols, and cholesterol through the blood. On the basis of surface tension and compressibility measurements of amphiphatic β-sheet consensus peptide of apoB8 and larger fragments of apoB9 it has been suggested that flexibility of the β-sheet motif assists in anchoring the whole apoB protein to lipoproteins in VLDL. Wingreen et al. analyzed the flexibility of R-helices and β-sheets by screening the structure of these motifs in crystalline databases. In the β-sheet they identified two dominant modes of twist and bend associated with the flexibility of the whole sheet.10 The macroscopic compressibility of a Langmuir monolayer is measured by the change in surface pressure, π, as a function of the mean molecular area, A, at the air-water interface, and it is defined as CM ) -∂(ln A)/∂π. In addition, the compressibility of the 2D crystalline assembly, CC, may be estimated based on shifts in the GIXD (0,1) Bragg peak position as a result of a change in surface pressure,4 CC ) -∂(ln d(0,1))/∂π. The peptide PFE-5 monolayer on the water surface exhibited three regions along the isotherm identified by distinct compressibility values. In the expanded state, at low surface pressure, π

10.1021/jp108496f  2011 American Chemical Society Published on Web 12/09/2010

Amphiphilic and Acidic β-Sheet Peptides < ∼1 mN/m, the monolayer exhibited high CM and CC values that are most probably associated with inter-ribbon adjustments, including molecular rearrangements occurring along the condensation of ordered domains that had formed spontaneously during film formation. Along the steep increase in surface pressure both the crystalline and monolayer compressibilities are small, whereas along the film collapse the crystalline compressibility CC remains low and that of the monolayer, CM, increases. Here we aimed at characterizing the effect the type of amino acid side chains may have on the crystalline compressibility of the β-sheet structure. We focused on a set of three amphiphilic and acidic β-sheet peptides having the general structure ProY-(Z-Y)5-Pro, with Y ) Asp (D) or Glu (E) and Z ) Phe (F) or Leu (L), denoted PFD-5, PLE-5, PLD-5, and the 1:1 molar ration mixture of PLD-5 and PLE-5. Monolayers of these peptides at the air-water interface were studied by GIXD along film compression and expansion. These three peptides and PFE-5 were recently shown to form in bulk, hydrogels composed of fibrils that are stabilized by hydrophobic interactions, and interstrand hydrogen bonds at near neutral pH values.6 Experimental Section Peptides were custom synthesized and purified by HPLC to 95% by Anaspec, CA. Monolayers were prepared by spreading a ∼0.1 mg/mL solution of each peptide, dissolved in trifluoroacetic acid/chloroform (1:9 v/v). Surface pressure-area isotherms of the monolayer films were measured using a KSV minitrough (KSV Instruments LTD, Helsinki, Finland). The mean molecular area is the area available on the Langmuir trough divided by the number of molecules spread. Noteworthy, the area per molecule measured in the π-A isotherm may be influenced by various factors including aggregation of the peptide in the stock solution used for spreading the film at the interface, peptide content in the lyophilized peptide stock powder used in the spreading solution, peptide purity, and its possible dissolution in the subphase. Consequently, the Langmuir isotherms of the peptides may show limiting areas per molecule that are smaller than those expected based on the assumed molecular projected area. GIXD experiments were performed with a liquid surface diffractometer at the BW1 undulator beamline at the HASYLAB synchrotron source (Hamburg, Germany). The peptide films were spread at room temperature, and diffraction measurements were performed at 5 °C. A monochromatic X-ray beam (λ ) 1.3039 Å) was adjusted to strike the liquid surface at an incident grazing angle Ri ≈ 0.85Rc (where Rc is the critical angle for total external reflection); this enhances surface sensitivity. The dimensions of the footprint of the incoming X-ray beam on the liquid surface were approximately 2 × 50 mm. GIXD signals were obtained from two-dimensional crystallites randomly oriented about the water surface normal. The scattered intensity was collected by means of a position-sensitive detector (PSD) which intercepts photons over the range 0.0 e qz e 1.3 Å-1, qz =2π/λ sin(Rf) being the out-of-plane component of the scattering vector and Rf being the exit vertical angle. Measurements were performed by scanning 2θxy, the angle between the projections onto the horizontal plane of the incident and diffracted beams; this then varies the horizontal component, qxy =2π/λ/(1 + cos2(Rf) - 2 cos(Rf)cos(2θxy))1/2 ≈ 4π/λ sin(2θxy) of the scattering vector. The diffraction data are represented in two ways: (1) The GIXD pattern I(qxy), obtained by integrating over the qz window of the PSD, shows Bragg peaks; (2) Bragg rod intensity profiles are the scattered intensities I(qz) recorded in

J. Phys. Chem. B, Vol. 115, No. 1, 2011 51

Figure 2. PFD-5 isotherms and diffraction patterns obtained along GIXD measurements. Letters assign the points at which the compression was halted and diffraction measurements were acquired. (A) Surface pressure versus area (π-A) isotherm; a-g, compression; g-i, expansion; and i-l second compression. The macroscopic compressibility, CM, m/N, values (underlined labels) were calculated based on the isotherm areas per molecule and the corresponding surface pressures for the first compression curve (see Experimental Section). (B) Selected d(0,1) Bragg peaks obtained along compression (line) and along expanded state and recompression, points h and on (dashed line). (C) Surface pressure versus ln d(0,1). The different regions along the curves are denoted by the calculated crystalline compressibilities, CC, m/N (underlined values). (D) Selected Bragg rods demonstrating shift in maxima along qz with the increase in surface pressure (arrow denotes the shift in Bragg rod maxima position).

channels along the PSD but integrated, after background subtraction, across the qxy range of each Bragg peak. The qxy positions of the Bragg peaks yield the lattice spacings d ) 2π/ qxy, which may be indexed by the two Miller indices h,k to yield the unit cell. The full-width at half-maximum (fwhm(qxy)) of the Bragg peaks yields the lateral 2D crystalline coherence length Lxy ≈ 0.9(2π)/fwhm(qxy). The width of the Bragg rod profile along qz gives a first estimate of the thickness of the crystalline film: hz ≈ 0.9(2π)/fwhm(qz). The diffraction data are represented as the intensities after correction for the Lorentz, polarization, and active area (LPA) factors. The π-A compression isotherms presented as π versus ln A may be fitted with linear equations. The inverse of the linear line slope is equal to the macroscopic compressibility of the monolayer, CM; for example, a slope of -87.9 mN/m corresponds to CM ) 1/(87.9 mN/m) × 1000(mN/1N) ) 11.4 m/N.4 Results The surface pressure area (π-A) isotherms of the Pro-Y-(ZY)5-Pro peptides exhibit in general three regions, the expanded, compressed, and collapsed states. These regions differ in their compressibility values, as demonstrated in Figure 2A for peptide PFD-5. The expanded state (Figure 2A, points a-d) extends down to the limiting area per molecule where the water interface is essentially fully covered by the peptide molecules. On further compression the film enters the compressed state that is characterized by a steep increase in surface pressure, followed by the collapse region, at π ≈ 24 mN/m (Figure 2A, d-f and f-g, respectively). GIXD measurements were performed along π-A compression (Figure 2A, a-g) and expansion (Figure 2A,

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Vaiser and Rapaport

TABLE 1: Summary of GIXD Data of PFD-5 (0,h) Peaks along the π-A Isotherm (Figure 2) point

π (mN/m)

mmaa (Å2/molecule)

qxy (Å-1)

d(0,1)b (Å)

fwhmxyc (Å-1)

Lxyd (Å)

Lxy/d(0,1)f

Ie (au)

A B C D E F G H I J K L

0 0.2 0.4 0.8 3.9 21.3 24.5 0.3 0.7 2 2.2 5.5

283 250 232 208 191 135 82 282 203 139 126 105

0.1397 0.1391 0.1426 0.1502 0.1593 0.1843 0.1852 0.1424

45.0 45.1 44.0 41.8 39.4 34.1 33.9 44.1

0.0255 0.0233 0.0217 0.0169 0.0149 0.0152 0.0208 0.0246

221 243 261 334 379 371 271 229

5 5 6 8 10 11 8 5

173 300 397 388 364 263 73 328

0.1038 0.1044

60.5 60.2

0.0298 0.0237

190 239

3 4

198 167

a Mean molecular area, measured along the isotherm. b The spacing corresponding to qxy, d ) 2π/qxy. c The full-width half-maximum of the Bragg peak at qxy. d The coherence length of the ordered domain Lxy ) 0.9 × 2π/fwhmxy. e The integrated intensity of the Bragg peak. f Lxy/d(0,1) provides a measure for the number of peptides ordered along the (0,1) long axis of the lattice.

g-i) isotherms. These curves display hysteresis with smaller nominal molecular areas along the expansion curve compared to the compression one. Indeed, a second compression of this film (Figure 2A, i-l) resembles the isotherm of the expansion curve. This hysteresis suggests that along the collapse peptide aggregates, probably in the form of multilayers, hence overall occupying a smaller area on the water surface. The PFD-5 Bragg peaks measured along the π-A isotherm, depicted in Figure 2B, were attributed to the repeat distance d(0,1) along the β-strand backbone axis (see Figure 1 and Table 1). The Bragg peak at d(0,1) ) 45.0 Å, which was obtained in the expanded state, at π ) 0 corresponds to the estimated length of a 13-residue peptide in the β-pleated conformation, as projected on the water interface that is ∼6.9/2 × 13 ) 44.8 Å (∼6.9 Å is the distance between every second amino acid in the pleated β-sheet conformation1). This spacing becomes smaller during film compression, as indicated by the shift in Bragg peak position to higher qxy values (lower d spacing). Up to points d and e (Figure 2B, Table 1) along the compressed state, the coherence length of the Bragg peaks increases, indicating growth in 2D of the ordered domains coherence length. At low surface pressure (Figure 2, point a) the coherence length of ordered domains is 221 Å, corresponding to ∼5 ordered strands along this direction, whereas at the beginning of the collapsed state, at point f, there are ∼11 ordered strands. Hence, the coherence length of the domains increases up to point e, with no significant change along the steep increase in surface pressure and decreases along the collapse, toward point g. At the collapse state (Figure 2B, f-g) the intensity of the d(0,1) peak sharply falls, indicating film destruction. On expansion to point i no peak was detected, but on further compression to point h (Figure 2B), still in the expanded state of the recompressed film, the peak reappears in the qxy position similar to that obtained at this area per molecule along the first compression (point c), demonstrating the elasticity of the crystalline β-sheet that was previously reported for the PFE-5 peptide of this family.4 The compressibility of the crystalline structure, along the d(0,1) direction, denoted CC, was calculated based on the shift in Bragg peak position with the applied surface pressure (see Experimental Section and calculated values underlined in Figure 2C). Up to the limiting area per molecule (Figure 2A, d), in the expanded state, the rather high compressibility CC ) 44.2 m/N is probably associated with molecular rearrangements within self-assembled domains that concomitantly also grow in size (see coherence lengths, Table 1). From point d to f, along the steep increase in surface pressure, in the compressed state of the film, the crystalline compressibility is CC ) 8.7 m/N. In

Figure 3. GIXD patterns of peptide PFD-5 obtained at the high qxy region. These peaks may be assigned to a subunit cell with average (see text) dimensions as ) 10.52, bs ) 6.45 Å, and R ) 108.9° generated by the pleated structure of the β-sheet assembly (Figure 4).

this region it is reasonable to assume that the strands within the lattice bend out of the interface (Figure 1), as evident by the shift in the Bragg rod qz maxima along d-g points (Figure 2D). These general trends in I(qxy) and I(qz) patterns have also been observed in the PFE-5 system4 and attributed to quasireversible, out-of-plane bending of the peptide backbone that yields to the lateral compression. Along the compressed state, from point f to g (Figure 2), the crystalline structure undergoes no further compression as indicated by f and g Bragg peaks remaining essentially in the same qxy position, however with a significant decrease in the intensity at from point f to g due to the large stresses exerted on the film. This same film was then again compressed (Figure 2A, points i-l), and interestingly, a different type of ordered assembly appeared with a single characteristic peak at a spacing ∼60 Å. This repeat distance is larger than the length of a single β-strand. The presence of the new diffraction peak and the absence of that assigned above as d(0,1) suggests formation of a new lattice at the expense of the former ordered phase. Alternatively, it is possible that the peptide film may exhibit two coexisting phases where at this point only this new phase was detected. On further compression to point l the Bragg peak of this new phase disappeared. In this new lattice there must be more than one molecule ordered along the (0,k) direction; however, with only one diffraction peak, we may only assume that at least two molecules generate the repeat order motif, giving rise to the 60 Å Bragg peak. Similar behavior with repeat distances that correspond to more than one molecule length were detected in the other peptides studies here and described below. Figure 3 shows three distinct Bragg peaks that were detected for PFD-5 monolayer at higher qxy values (Table 2) which unlike the (0,1) peaks hardly shifted with the increase in surface

Amphiphilic and Acidic β-Sheet Peptides

J. Phys. Chem. B, Vol. 115, No. 1, 2011 53

TABLE 2: Summary of GIXD Data and PFD-5 Peaks at the High qxy Region (Figure 3 and π-A isotherm in Figure 2A) π (mN/m)

mmaa (Å2/molecule)

qxy (Å-1)

db (Å)

(h,k)sc

fwhmxyd (Å-1)

Lxye (Å)

If (au)

unit cellg

a

0.0

282

0.3

250

c

0.5

232

d

0.9

208

e

4.0

191

f

20.3

135

g

25.3

82

k l

2.2 5.5

126 105

6.07 5.02 4.67 6.10 4.98 4.67 6.15 4.95 4.67 6.13 4.95 4.66 4.95 4.65 4.94 4.62 4.92 4.44 4.76 4.77

(0,1) (2,0) (2,-1) (0,1) (2,0) (2,-1) (0,1) (2,0) (2,-1) (0,1) (2,0) (2,-1) (2,0) (2,-1) (2,0) (2,-1) (2,0) (2,-1) (2,0) (2,0)

0.0597 0.0656 0.0519 0.0926 0.0579 0.0860 0.2543 0.0424 0.0597 0.2492 0.0411 0.0446 0.0360 0.0352 0.0528 0.0422 0.0708 0.0594 0.0513 0.0431

95 86 109 61 98 66 22 133 95 23 138 127 157 161 107 134 80 95 110 131

1445 680 904 1694 467 1186 1552 579 1108 2221 690 973 428 442 382 281 135 114 564 631

a ) 10.60 Å, b ) 6.40 Å, γ ) 108.6°

b

1.0356 1.2512 1.3458 1.0299 1.2609 1.3467 1.0219 1.2703 1.3466 1.025 1.2693 1.349 1.2698 1.3523 1.2708 1.3606 1.2764 1.4162 1.3198 1.3172

a ) 10.52 Å, b ) 6.44 Å, γ ) 108.8° a ) 10.46 Å, b ) 6.50 Å, γ ) 109.0° a ) 10.46 Å, b ) 6.48 Å, γ ) 108.9°

a Mean molecular area, measured along the isotherm. b The spacing corresponding to qxy, d ) 2π/qxy. c Miller indices assigned to a subunit cell according to the three Bragg peaks obtained at this area per molecule. d The full-width half-maximum of the Bragg peak at qxy. e The coherence length of the ordered domain Lxy ) 0.9 × 2π/fwhmxy. f The integrated intensity of the Bragg peak. g The subunit-cell dimensions, assigned to the three Bragg peaks observed at this area per molecule.

Figure 4. β-Sheet subunit-cell assembly demonstrated here with a sequence of Gly residues for clarity. The strands are characterized by φ ) -113, Ψ ) 113, and the donor-acceptor hydrogen-bond distance is ∼2.8 Å. (A) Top view along the normal to the interface showing two neighboring strands in the antiparallel mode. The bottom dashed line denotes the (2,0) axis of the subunit cell. (B) View along the (2,0) spacing direction showing the offset of ∼1.7 Å between neighboring strands.

pressure. These Bragg peaks (Figure 3 and Table 2, a-d) that correspond to the average spacing values 6.11, 4.98, and 4.67 Å may be assigned to a subunit cell as ) 10.52 Å, bs ) 6.45 Å, and R ) 108.9° that is generated by the pleated structure of the β-sheet assembly (Figure 4). Along the as axis there are two neighboring strands packed in the antiparallel mode,1 and the bs is defined by the pleated strand motif. In this subunit cell the area per residue is 16.1 Å2, and the observed repeat distances are indexed ds(0,1), ds(2,0), and ds(2,-1). Interestingly, this is the first diffraction data which provides evidence for the pleated

β-sheet structure at interfaces, detected by GIXD measurements. This subunit cell indicates that neighboring strands are offset by as/2 × cos 71.1 ) 1.7 Å. Noteworthy, two of these three peaks (4.98 and 4.67 Å) were previously observed in GIXD measurements of the peptide (Phe-Glu)5-Phe monolayer at the air-water interface that formed assemblies ordered predominantly only along the interstrand hydrogen-bond direction.1 Due to the lack of additional data these peaks were then assigned to higher order spacings of the peptide lattice. Nevertheless, these same reflections were also observed in this study for PLD-5, described below. Hence, it may be concluded that these Bragg reflections indeed correspond to a common motif to β-sheet structures. This subunit cell is probably generated by a preferred apposition of strands in β-sheet structures with a hydrogenbond pattern, depicted in Figure 4. This interstrand arrangement resists the compression and shows only minor differences in Bragg peak position as a function of surface pressure (Table 2). These minor changes in peak position are accompanied by weakening of the ds(0,1) Bragg peak with compression to complete disappearance at π ) 4.0 mN/m, where the β-strands deform significantly as a result of the applied surface pressure. In addition, ds(2,0) spacing becomes only slightly shorter (Figure 3, points a-g and Table 2) from 5.02 to 4.92 Å with essentially no change in the (2,-1) spacing. The high resistance to compression this subunit cell presents may be the result of the tight molecular packing (Figure 4). The appearance of a single Bragg peak in this qxy region, at points k and l (Table 2, Figure 3) corroborates with the detection of d(0,1) ≈ 60 (points j-k, Table 1) mentioned above, indicative of the appearance of a new phase along the recompression described above. Peptide PLD-5, the most hydrophilic peptide among those studied herein, exhibits, in the expanded state, an ordered structure with a repeat distance d(0,1) ) 84.9 Å (Figure 5A and 5B and Table S1, Supporting Information), obviously almost as large as the length of two strands. A spacing larger in length than the peptide long axis was detected for peptide PFD-5 but only after film collapse. Beyond the steep increase in surface pressure at π ) 4.6 mN/m in the compressed state (Figure 5, e-k), the d(0,1) spacing is smaller than ∼45 Å, suggesting the

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J. Phys. Chem. B, Vol. 115, No. 1, 2011

Figure 5. PLD-5 isotherms and diffraction patterns. Letters assign the points at which film compression was stopped and diffraction measurements were acquired. (A) π-A isotherm. Underlined values are calculated macroscopic compressibility, CM, m/N (see Experimental Section). (B) GIXD Bragg peaks that correspond to the spacing along the peptides’ long axes. Bragg peaks obtained along expansion of the film are shown by the dashed line. (C) Surface pressure versus ln d. Underlined values are the calculated crystalline compressibilities, CC, m/N (see Experimental Section). Points a and b marked by asterisks denote very weak intensities; hence, the exact position fits are less reliable. (D) GIXD (patterns at high qxy values which in the compressed state (f-h and possibly k)) may be assigned to the subunit cell shown in Figure 4.

appearance of crystalline phase with one peptide along the (0,1) axis. This phase is characterized by higher peak intensities (Figure 5B, e-g) and larger coherence lengths (Table S1, Supporting Information). In the compressed state PLD-5 ordered domains react similarly to PFD-5 and PFE-54 by shifting the Bragg peaks to lower qxy values (Figure 5B) and the rods’ maxima to higher qz values (data not shown). The crystalline compressibility in the compressed state CC ) 5.8 N/m (Figure 5C) is rather lower as shall be further discussed. The diffraction pattern of PLD-5 at high qxy values, though rather weak, shows in the expanded state (Figure 5D, point c) one Bragg peak corresponding to d(2,0) ) 4.80 Å (Table S2, Supporting Information), whereas along the steep increase in surface pressure, the two typical peaks of the subunit cell described above appeared (Figure 5D and Table S2, Supporting Information, points e-h). Noteworthy, this subunit cell lattice forms here in the compressed state, in agreement with the close-packing characteristics attributed to this motif in PFD-5. Close to the collapse state (beyond point k) and along film expansion (points l-n) only the single d(2,0) peak appears at 4.68-4.78 Å (Table S2, Supporting Information). The peptide PLE-5 showed a Bragg peak in the expanded state (Figure 6, points a-c) at d spacings corresponding to 52.9-44.2 Å (Table S3, Supporting Information) close in value to the length of the peptide. In the compressed state (Figure 6, points d-j) peak intensities in general increase along with a small decrease in the corresponding coherence lengths (Table S3, Supporting Information). The crystalline compressibility in the compressed state (Figure 6C) was found to be CC ) 8.1 m/N. PLE-5 exhibited a d(2,0) Bragg peak at 4.80-4.74 Å all along the isotherm (Figure 6D and Table S4, Supporting Information). GIXD measurements were also performed on a monolayer composed of a 1:1 molar ratio mixture of peptide PLE-5 and

Vaiser and Rapaport

Figure 6. PLE-5 isotherms and diffraction patterns. Letters assign the points at which film compression was stopped and diffraction measurements were acquired. (A) π-A isotherm. Underlined values are calculated macroscopic compressibility, CM, m/N (see Experimental Section). (B) GIXD Bragg peaks that correspond to the spacing along the peptides’ long axes. Bragg peaks obtained along expansion of the film are shown by the dashed line. (C) Surface pressure versus ln d. Underlined values are the calculated crystalline compressibilities, CC, m/N (see Experimental Section). (D) GIXD patterns at the high qxy region.

Figure 7. PLD-5:PLE-5 (1:1 molar ratio) monolayer. Letters assign the points at which film compression was stopped and diffraction measurements were acquired. (A) π-A isotherm. Underlined values are calculated macroscopic compressibility, CM, m/N (see Experimental Section). (B) GIXD Bragg peaks that correspond to the spacing along the peptides’ long axes. (D) Selected Bragg rods of the corresponding peaks demonstrating shift in maxima with the increase in surface pressure.

PLD-5 (Figure 7). It appeared in general to form a homogeneous well-mixed film with no evidence in diffraction pattern for phase separation. The mixture formed in the expanded state an ordered structure with d(0,1) repeat distances ∼70 Å (Table S5, Supporting Information) larger than the length of the peptides. This phase then transformed, in the compressed state (Figure 7, points e-h), into the phase that is characterized by spacing corresponding to the peptide length. Interestingly, the Bragg rod of

Amphiphilic and Acidic β-Sheet Peptides

Figure 8. (A) Coherence length and (B) detected d(0,1) spacings of the peptide monolayers versus surface pressure: PFD-5 (squares), PLE-5 (solid triangles), PLD-5 (solid circles), and PLD-5:PLE-5 (triangles). Lines added to guide the eye.

the (0,1) peak shifted its maxima with the increase in surface pressure (Figure 7D), similar to the behavior of the other pure peptide monolayers. Only at one point f along the measurements a Bragg peak (not shown) corresponding to the hydrogen spacing d ≈ 4.65 Å was detected. This result may be, in principle, interpreted as indicative of disorder along the interstrand hydrogen-bond direction. However, the crystalline compressibility of the mixed film was relatively low, CC ) 6.1 m/N (Figure 7C) similar to that of PLD-5 (5.8 m/N), pointing to a relatively stable crystalline phase. These results which may appear to be contradictory can be rationalize by assuming formation of nonisotropic crystallites, where in this case it is reasonable that crystallites would be elongated in the (2,0) direction. GIXD measurements in the configuration used in this study (which is suitable to two-dimensional powder, see the Experimental Section) may miss crystallites that are nonisotropically distributed, hence showing no diffraction. An indication that supports the assumption that crystallites did form in the mixed film manifested in the relative high stability of the mixed film that showed coherence lengths along the (0,1) direction (Table S5, Supporting Information) larger than those detected for the other peptides despite the fact that this is a mixed film. Summary and Discussion This study was motivated by our interest in evaluating the effect of amino acid side chains on the compressibility of crystalline β-sheet structures at interfaces. We focused on a family of amphiphilic and acidic β-sheet peptides of the same length forming monolayers at the air-water interface. The stability of these films is supposed to be governed by intermolecular interactions that are hydrophobic on one face of the sheet and mainly based on hydrogen bonds on the opposite face of the sheet. The isotherms of these peptides exhibit three regions, the expanded, compressed, and collapsed states, that are clearly distinguished by large differences in the values of their crystalline compressibilities, CC, with the largest one observed in the expanded state and the smallest in the collapsed state. In the expanded state that reaches a surface pressure ≈ 1 mN/m, crystalline domains are already present. Along further compression these domains generally grow in size as evident in Bragg peaks coherence lengths (Figure 8A). The ordered domains that exist in the expanded state do not cover the whole area of the trough yet; during film compression there is both an increase in size of the ordered domains accompanied by a decrease in the lattice d(0,1) spacing (Figure 8B). The decrease in the lattice spacing is most probably accompanied by interstrand rearrangements. On crossing the limiting area per molecule, the steep increase in surface pressure arises from the resistance of the closely packed molecules to the external stresses. Along the compressed state the d(0,1) spacing becomes smaller due to

J. Phys. Chem. B, Vol. 115, No. 1, 2011 55 bending of the backbone as evident by a shift in the (0,1) Bragg rods maxima toward higher qz values. Following expansion and release of surface pressure the peptides revert to the d(0,1) spacing obtained at the same surface pressure along the compression, indicating the elastic nature of the backbone deformation. In the collapsed state, along the region in which there is little change in surface pressure, the crystalline domains maintain the deformed conformation with the short d(0,1) spacing (Figure 8B). The coherence lengths of the diffracting domains (Figure 8A) increase in general up to either the end of the expanded state at π ≈ 1 mN/m (PLE-5) or the compressed state at π ≈ 10-17 mN/m (the other peptides). A decrease in domain size may occur along the compressed state and continue in the collapse state. The compressibility values for this group of peptide were found to vary between 8.7 and 5.8 m/N. We were interested to learn whether the compressibility may also be influenced by impurities as peptides samples were purified to >95% by HPLC (see Experimental Section) and that may affect the quality of the two-dimensional crystals. PFD-5 of a different synthesis batch was also studied (data not shown) and found to have 7.6 m/N compressibility. This result suggests that the crystalline compressibilities of three of the peptides PFD-5, PFE-5,4 and PLE-5, 8.7, 7.4, and 8.1 m/N, respectively, exhibit in general similar compressibility values, in context of their