Conformational Study and Hydrogen Bonds Detection on Elastin

Biomacromolecules 2005, 6, 1299-1309

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Conformational Study and Hydrogen Bonds Detection on Elastin-Related Polypeptides Using X-ray Photoelectron Spectroscopy Roberta Flamia,† Giuseppe Lanza,† Anna M. Salvi,*,† James E. Castle,‡ and Antonio M. Tamburro† Dipartimento di Chimica, Universita` della Basilicata, via N. Sauro 85. 85100 Potenza, Italy, and School of Engineering, University of Surrey, Guildford GU2 7XH, Surrey, United Kingdom Received November 8, 2004; Revised Manuscript Received February 15, 2005

The chemical bonds of the pentapeptide sequence of elastin ValGlyGlyValGly (VGGVG), both in its monomer and polymer forms, were correlated with their XPS spectra through a well-established curve-fitting procedure. To aid in this correlation, the C1s, O1s, and N1s chemical shifts of the Boc-VGGVG-OEt, were validated by theoretical calculations, performed in the framework of the Koopman approximation of HF/6-31G molecular orbitals, leading to the “preferred” conformation of the protected monomer. Then the same curvefitting procedure was adopted for interpreting the XPS spectra of the polypentapeptide as a powder, and the XPS results obtained both for monomer and polymer compounds were compared with those obtained by FT-IR. The polymer was then analyzed after deposition onto a silicon substrate, Si(100), either from methanol or water suspensions and the presence of hydrogen bonds was detected at the polymer/substrate interface and between the polymer chains. The “surface rearrangement” that could be inferred from XPS results strongly confirms that derived from AFM images previously obtained under the same experimental conditions. In particular, the observed amyloid conformation is stabilized by hydrogen bonds to water molecules included in the structure while the formation of the beaded string structure observed in deposits from methanolic suspension is probably mediated by hydrogen bonds to the hydrated silicon surface. Introduction Elastin, the protein that provides elasticity in vertebrates, has a peculiar amino acid composition characterized by repetitive sequences rich in hydrophobic residues.1 The most frequently occurring is a general one of type “XGlyGlyZGly” (X, Z ) Val, Leu, or Ala). Many sequential polypeptides of this type have been studied, extensively, by Tamburro and co-workers.2-4 In particular, previous microscopy studies performed on the polypentapeptide, poly(VGGVG), have highlighted many supramolecular features that are characteristic of the parent protein, Elastin.5 Furthermore, the polymer, imaged by atomic force microscopy (AFM) under ambient conditions, showed a dynamic behavior that resulted from surface energy considerations in relation to the characteristics of the supporting medium.6 Finally, the presence of amyloid-like fibers was revealed by both TEM and AFM. In undertaking the present study, it was anticipated that considerable advances in understanding the mechanism by which supra-molecular structures are developed could be made by correlating morphological imaging by AFM with the information on chemical state obtained by XPS. This correlation has been assisted and amplified by use of molecular modeling of the pentapeptide monomeric unit. The surface-specific technique, X-ray photoelectron spectroscopy * To whom correspondence should be addressed. E-mail: [email protected]. † Universita ` della Basilicata. ‡ University of Surrey.

(XPS), has an almost unique ability to investigate the chemical composition within a limited depth of analysis.7 Thus, although studies by AFM of such biomacromolecules on surfaces can reveal distinct morphologies according to the polarity of the substrate on which they are deposited and depending on the suspending medium used to distribute them on the surface, XPS can be used to reveal the outermost chemical groups associated with these distinctive morphologies. The effects of conformation and orientation of the biopolymer will be small but could be observable with XPS, particularly if high performance equipment is used.8 However, as shown in this paper, when the XPS spectra are analyzed using an accurate curve fitting procedure, the detection of hydrogen bonding associated with the geometrical rearrangements of the molecular films9 is possible, even using conventional equipment. The experimental work was subdivided into the following steps: 1. XPS characterization of the protected monomer, BocVGGVG-OEt, and parallel theoretical analysis of its core electron structure to be used as a guideline at a qualitative level for the interpretation of the C1s, O1s, and N1s curvefitted results. 2. XPS characterization of poly(VGGVG) using the same curve-fitting procedure, before and after deposition onto a silicon surface according to the procedures previously adopted for AFM.6

10.1021/bm049290s CCC: $30.25 © 2005 American Chemical Society Published on Web 03/17/2005

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3. Comparison of peak assignments on XPS and FT-IR spectra, both for monomeric, Boc-VGGVG-OEt, and poly(VGGVG). Experimental Section XPS and Materials. The XPS spectra were acquired with a LH X1 Leybold instrument using unmonochromatized Al KR (1486.6 eV) operating at a constant power of 260 W. Wide and detailed spectra were collected using the FAT (fixed analyzer transmission) mode of operation with a pass energy of 50 eV and a channel width of 1.0 and 0.1 eV, respectively. Under these conditions, the instrumental contribution to line-width is kept constant and the measured fwhms (full width half maximum) of Au 4f7/2 (84.0 eV) and Cu2p3/2 (932.7 eV) signals used for calibration purposes were 1.3 and 1.6 eV, respectively. The synthesis of both the monomer and the polymer has been described elsewhere.6 Boc-VGGVG-OEt and poly(VGGVG) compounds were in the form of dry powders and were mounted on the sample holder by pressing them on a double adhesive copper tape. The samples were then transferred to the analysis chamber where the vacuum was always better than 5 × 10-9 mbar. Care was taken to be sure that no signals from the adhesive tape were visible on the wide spectra. The polymer was also analyzed deposited on a “pretreated” Si(100) surface from both a methanolic and an aqueous suspension. The substrate pretreatment simply consisted of washing it with the same solvent used for the polymer suspension: the silicon substrate was then exposed to XPS analysis to verify the status of its surface before the polymer deposition. Curve-Fitting Program. The acquired XPS spectra were subsequently analyzed using a curve-fitting program, Googly, that was fully described in previous work10,11 and proposed as an alternative method to the standard Shirley procedure.12 Googly allows the simultaneous fitting of the photopeaks in the form of a Voigt function and their associated background, in a wide energy range, thus retaining information from both intrinsic and extrinsic processes associated with photoemission.10,11 Peak areas were converted to composition in At% using established procedures and the appropriate sensitivity factors (SF).13,14 The potentiality of the Googly program was here limited to the peak region and various constraints were applied to reduce the free parameters: for example, unless otherwise specified, the peak shapes and fwhms were forced to be the same for the given core line envelope and the relative intensities derived from the molecular formulas were used as starting guesses for the component peaks. When necessary, some constraints were released leaving the program to reach the convergence and the criterion then adopted, in reaching a decision on the best fit, was that the fitting results with correctly chosen peak and background parameters should give the elemental mass balance which reflects the stoichiometry of the given molecular formula, in the limit of XPS accuracy.15,16 The XPS figures reported in this paper are not corrected for surface charging but the peak assignments (Binding Energies, BEs), as reported in the tables, are referenced to

Figure 1. Ground-state molecular structure of the Boc-VGGVG-OEt peptide.

C1s aliphatic carbon, as an internal standard, set at 285.0 eV and to literature data (See NIST database:www.nist.gov). The widescan spectra are reported, as acquired, in kinetic energy, whereas the energy scales of the detailed regions are converted to binding energy so as to facilitate comparison of the curve fitted results with literature data. Electronic Structure Calculations. Theoretical analysis of the core-electron structure has been performed in the framework of the Koopman approximation of HF/6-31G molecular orbitals.17 In this approach, the relaxation of molecular orbitals and the correlation energy variation, occurring upon ionization of one electron from the closedshell structure, are not considered thus hampering a quantitative theoretical/experimental comparison. However, the core nature of the C1s, N1s, and O1s suggests that relaxation and correlation effects should be constant for each class of molecular orbitals.18 In addition, electron-releasing and electron-withdrawing effects are explicitly included without adoption of any modeling of the real molecule. Therefore, the interpretation of experimental XPS data of protected BocVGGVG-OEt pentapeptide was based, at a qualitative level, on the present theoretical data. FT-IR. Fourier transform infrared spectra were obtained in transmission on a Jasco FT/IR 460 spectrophotometer by using a resolution of 4 cm-1 and 200 scans accumulation and then smoothed with the Savintky-Goolay algorithm. The samples were examined in the solid state, as KBr pellets. Results Core-Electron Structure and XPS Curve-Fitting of Boc-VGGVG-OEt. The forerunner of the synthetic polymer poly(VGGVG) is the protected monomer Boc-VGGVG-OEt. Current studies of this monomer, that include FT-IR, CD, 1H NMR spectra, and ab initio calculations,19 have shown that it is preferably folded (β-turn like, type II) and stabilized by a C14 pseudocycle involving a hydrogen bond between the NH group of the second residue (glycine) and the CdO of the terminal ester functional group. In the molecule, there are two additional hydrogen bonds, weaker than the first one, which contribute to form the C7, γ-turn structures reported in Figure 1. Such a conformation was reported to be the one found experimentally for similar monomers in previous studies.20 XPS data are then compared and interpreted on the basis of the theoretical data derived for this conformer. Figure 2a shows the monomer wide scan, demonstrating the absence of foreign elements, and its molecular formula.

On Elastin-Related Polypeptides Using XPS

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Figure 2. Wide scan of (a) the protected pentapeptide Boc-ValGlyGlyValGly-OEt and (b) the polypeptide poly(ValGlyGlyValGly), and related molecular formulas. The main photoelectron C1s, N1s, and O1s peaks and their associated X-ray excited Auger peaks, CAES, OAES, and NAES are labeled. The Auger peaks, not investigated in this article, are broad and contribute little to chemical state information.

Quantitative XPS analysis of the sample gives the relative ratio of the C:N:O signals as 4.2:1.0:1.6, compared with the stoichiometric ratio of the three elements, as derived from the molecular formula, of 4.6:1.0:1.6. The reason for the small carbon defect (8.7%) has not been investigated considering that it remains within the total experimental error ((10%).15,16 Theoretical data show three different groups of orbitals with an ionization energy “centred” at approximately ∼307.5, ∼559.3, and ∼425.5 eV; associated with the C1s, O1s, and N1s orbitals, respectively, Figures 3 and 4. As mentioned above, without explicitly considering the huge relaxation and correlation energies, the energy ranges of these groups of orbitals can only be in qualitative agreement with the binding energies from XPS. However, the energy distributions assigned to different chemical states can be compared, for the same orbital, with those obtained by curve-fitting the relevant detailed region (vide infra). C1s Region. The C1s orbitals can be grouped into four well-separated sets because of the different electronic properties of the atoms directly bonded to carbon atoms, as seen in Figure 3. At low ionization energy there are 10 1s orbitals of aliphatic carbons of ethyl, iso-propyl, and tert-butyl groups. These orbitals spread over 1.34 eV and roughly they have a symmetric distribution around the energetic baricenter; therefore, they should appear as one envelope in the XPS spectrum. At 1.01 eV higher ionization energy, there is a set of seven orbitals associated with the C1s of atoms linked through a single bond to oxygen or nitrogen, i.e., the R-carbon and the terminal -O-C- ester and urethane groups. These orbitals spread over 1.01 eV and roughly have a symmetric

Figure 3. Ionization energy, in the Koopman approximation, of C1s orbitals for the ground-state structure of the Boc-VGGVG-OEt peptide. The “/” indicates energy levels of carbonyl groups involved in hydrogen bonding.

distribution around the energetic baricenter; therefore, they should appear as a single envelope in the XPS spectrum.

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Figure 4. Ionization energy, in the Koopman approximation, of O1s and N1s electrons for the ground-state structure of the Boc-VGGVGOEt peptide. The “/” indicates energy levels of >CO and >NH groups involved in hydrogen bonding.

The four peptidic carbonyl C1s orbitals are separated into two couples. In fact, the presence of electron-releasing isopropyl groups in the R-carbon lowers the >CO ionization energy of the Val units. However, the energy separation (0.52 eV) is not enough to be clearly separated into two components in the XPS spectrum and a four-components signal is more appropriate. The C1s orbitals at highest ionization energy are those of the protective ester and urethane functionalities. In these cases, the carbons are directly bonded to two strong electronegative oxygen atoms. These two orbitals are sufficiently separated, energetically (0.76 eV), from the four peptidic carbonyls and should consist of a single signal in the XPS spectrum. It is important to note that the C1s ionization energy of the peptidic >CO groups involved in hydrogen bonding lies at higher ionization energy (they are indicated by “/” in Figure 3). The curve-fitted C1s region is shown in Figure 5a, whereas Table 1 reports the fitting parameters. The carbon signal was fitted with five peaks. The first one, “peak 0”, was recognizable also in the spectra of analogous samples, and it is assigned to small quantities of solvent trapped in the powder during synthesis and the purification processes or to contaminants of a different nature. Peak 1 was placed at 285.0 eV in Table 1 and used for the determination of the sample surface charging (2.5 eV). This signal is due to emission from “aliphatic” carbons (that are bonded with single bonds to hydrogen or other carbon atoms) of both the valine residues and terminal protector groups.

Flamia et al.

Peak 2 contains the signal of the carbons in the R position in the five peptidic residues and of the carbons bonded with a single bond to an oxygen atom in the two terminal groups. Peak 3 deals with the four peptidic carbonyls. Peak 4 contains the signals of the carboxylic carbons of both ester and urethane terminal groups. O1s Region. Theoretical data show five O1s orbitals (distributed over 0.75 eV) at low ionization energy, Figure 4 (upper part). They are associated with the four carbonyl groups of peptide and the one due to the protective Boc group. The carbonyl group of the -COOCH2CH3 terminal ester and the two -O- atoms of both the protective ester and urethane lie at higher ionization energy. Among the >CO groups, those involved in hydrogen bonding lie at higher ionization energy. The energy separations among the various O1s orbitals suggest a 5:1:1:1 distribution of the XPS peaks. The XPS O1s signal was well fitted with two components with an area ratio of 5 to 3 and with their fwhms as free parameters (Figure 5b and Table 1). In accord with the calculations, peak 1 deals with the four oxygens present in the pentapeptidic chain plus the carbonyl oxygen of the Boc protector group. The broader peak 2, contains the signal from the remaining carbonyl oxygen of the terminal ester and the -O- signals of both terminal protecting groups. As is evident from Figure 4, these last three signals should be well spaced in energy (∼0.5 eV, each), and, in fact, the fwhm of peak 2 was required to be proportionally larger than peak 1 (Table 1). N1s Region. The five 1s orbitals of the >NH groups spread in a narrow energy range (0.94 eV, Figure 4, lower part) because of the great electronic similarity of the two bonded units, C(carbonyl) and C(aliphatic). Therefore, a single peak might be proposed for the XPS signal at 400 eV. However, it should be noted that the N1s orbitals of NH groups involving hydrogen bonding lie at lower ionization energy than the free NH group. This produces an asymmetric energy distribution of the various N1s orbitals thus suggesting a slightly asymmetric XPS peak. The XPS nitrogen signal appears as a single peak, centered at 400.0 eV, with a fwhm of 2.33 eV (Table 1) that is compatible with the predicted energy separation among the five orbitals (0.94 eV). The different signals are not distinguishable, because the five nitrogen atoms present in the molecule are too close in energy for our spectrometer’s resolution. However, the need for an extra contribution to account for the slight asymmetry, at the lower BE side of the peak, is evident, looking at the excursion in the residuals plotted at the top of Figure 5c. An alternative fit was performed, using two peak components of narrower fwhms and different relative intensities with the result that the overall N1s area was practically the same. Therefore, the single peak reported in Figure 5c was considered acceptable to account for the contribution of the five N1s orbitals. The above results indicate that, for each orbital examined, the area ratio of the peak components, reported in Table 1, is in good agreement with theoretical analysis, on the scale of our instrumental resolution. As described in the next paragraphs, the curve fitting procedure was then used to



Figure 5. Curve fitting for Boc-ValGlyGlyValGly-OEt, in the (a) C1s, (b) O1s, and (c) N1s regions. The solid line represents results of fitting, whereas the points represent the experimental data. In the upper part of the figure, the difference between the fitting results and the experimental points is shown. Table 1. Boc-ValGlyGlyValGly-OEt Curve Fitting Parameters

Table 2. Curve Fitting Parameters for Poly(ValGlyGlyValGly)

corrected area

BE

corrected normalized

area

BE

area

normalized

peak

corr.

fwhm

(arbitrary

area

orbital

no.

(eV)

(eV)

units)

ratios

assignment

contam. C (C,H) CR, C-O N-C)O O-C)O C)O Boc-O-C, COOEt N-H

C1s

interpret the XPS spectra of the same pentapeptide sequence when polymerized and when interacting with different media. Powdered Polymer. Characterization of poly(VGGVG) by MALDI mass spectrometry (data not shown) has ascertained that the number of pentameric units was in the range 2-9, corresponding to a molecular mass range of 775-3500 Da, having a pseudo-log-normal distribution. It was also shown that its N-terminal is likely to be formylated. From the distribution of the pentameric units number-centered on 5, it is possible to estimate the contribution of the terminal groups to the photoelectron signals associated with the polymer repeat unit. The nitrogen atom placed in the terminal position (4% of the total N1s signal) is not distinguishable from the nitrogen atoms belonging to the polypeptidic chain, whereas in the case of the C1s region, this contribution is only 1.2% of the total signal and, therefore, hardly discernible by curve fitting. In the case of the O1s region, however, the contribution from the terminal groups (reported as 〈Tg〉 in Table 2) is around 7.4% of the total signal and accounts for the two additional peaks at both ends of the main O1s peaks (see Figure 6b). The Tg contributions should not be included when deriving the signal ratio of the polymer repeat unit. Making this correction, the quantitative analysis gives a C:N:O signals ratio of 3.2:1.0:1.0 for the sample, in perfect agreement with that derived from the polymer stoichiometry (Figure 2b). As with the monomer, the wide scan of the polymer (Figure 2b) does not show any elements other than C, N,

N1s

0 1 2 3 4 1 2 3 4 1 2

283.2 285.0 286.2 287.9 288.9 529.4 531.5 532.4 534.0 399.3 400.2

1.97 1.97 1.97 1.97 1.97 2.10 2.10 2.10 2.10 2.13 2.13

126.47 903.79 753.16 600.22 162.06 31.92 600.61 162.17 31.92 166.65 595.87

0.9 6.0 5.0 4.0 1.1 0.2 3.9 1.1 0.2 1.1 3.9

contam. C (C,H) CR C)O C)O‚‚‚H C)O Tg C)O C)O‚‚‚H COOH Tg N-H‚‚‚O N-H

peak

corr.

fwhm (arbitrary

orbital

no.

(eV)

(eV)

units)

ratios

assignment

C1s

0 1 2 3 4 1 2 1

282.7 285.0 286.5 288.2 289.5 531.6 533.3 400.0

2.09 2.09 2.09 2.09 2.09 2.26 2.56 2.33

92.29 1946.10 1362.27 815.38 407.69 1074.08 644.45 1073.09

0.5 9.9 6.9 4.1 2.1 5.0 3.0 5.0

O1s N1s

O1s

and O. The fitting parameters of the C1s, O1s, and N1s regions are listed in Table 2. C1s Region. The C1s spectrum is shown in Figure 6a. It is possible to recognize five different components. Peak 0 has the same meaning as indicated for the monomer, i.e., trapped solvent. In this case, the polypeptide was purified by several cycles in which it was stirred in ethanol for about 2 h, then filtered, and left to dry at ambient temperature. Peak 0, which initially contributed 25% of the total C1s signal, gradually reduced to 5% without affecting the relative area ratio of the other peak components. The component indicated as peak 1, placed at 285.0 eV and used as reference for the sample surface charging calculation (that is 2.2 eV), is assigned to aliphatic carbons of the valine lateral chains in the pentameric unit. Peak 2 arises from the photoelectron emission of the five R-carbons of the repeating units. The components indicated as peaks 3 and 4, separated by 1.0 eV, were assigned to carbonylic carbons in the polypeptide chain, the oxygen of which is involved (peak 4) or not (peak 3) in the formation of a hydrogen bond. This hypothesis seems to be confirmed by the O1s and N1s spectra: in both regions two components are evident with an area ratio comparable with that of peaks 3 and 4. The carbon signals from the C-terminal carboxylic and N-terminal formyl-groups are not discernible, and should

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Figure 6. Curve fitting for poly(ValGlyGlyValGly) in the (a) C1s, (b) O1s, and (c) N1s regions. See also caption of Figure 5.

they superimpose those belonging to the polypeptidic chain, they would have practically no influence on the relative peaks intensity, as explained above. O1s Region. Figure 6b shows the oxygen 1s spectrum. Peaks 2 and 3 are assigned to carbonylic oxygen of the polypeptidic chain, involved (peak 3) or not involved (peak 2) in the formation of a hydrogen bond. The relative normalized areas of the two components and their energy separation, 0.9 eV, are comparable with those of the corresponding carbon signals (peaks 3 and 4 in the C1s region). As said above, we have supposed that peak components 1 and 4 derive from the emission of the C-terminal group. In particular, we have tentatively assigned peak 1 to the oxygen double bonded to carbon, and peak 4 to the oxygen, O-H, of the carboxylic group. N1s Region. Figure 6c shows the nitrogen 1s spectrum. As for the monomer, a slightly asymmetric shape is still evident, but in this case, the main contributions derive from two nitrogen components related to the emission of atoms involved (peak 1) or not involved (peak 2) in the formation of hydrogen bond. Their relative areas and binding energy difference are, in fact, comparable with the corresponding carbon and oxygen signals. The curve fitting results shown in Table 2 summarize the analysis reported above for each orbital examined: “strong” hydrogen bonds, not detected on the monomer, are now evident among the polymer chains. The presence of strong and weak hydrogen bonds can also be deduced from the FT-IR spectra reported in Figure 7, where the monomer and the polymer absorbance are superimposed in the frequency range of 1500-1800 cm-1. The two spectra are qualitatively comparable, considering the presence in the monomer spectrum of the Boc (urethane) and OEt (ester) absorptions, which are easily recognizable in the figure. A qualitative comparison between XPS and FT-IR data can be made and related, also, to the computational results. In fact, the XPS chemical shifts of the >CO and >NH groups involved or not involved in hydrogen bonding have found a counterpart in the theoretical results of Boc-VGGVG-OEt and FT-IR data of both Boc-VGGVG-OEt and poly(VGGVG). Computational data suggest an increase of both C1s and O1s ionization energies of the peptidic groups involved in hydrogen

Figure 7. FT-IR spectra of Boc-VGGVG-OEt (solid line) and poly(VGGVG) (dashed line) in the amide I (1800-1600 cm-1) and amide II (1600-1500 cm-1) regions.

bonding and, conversely, a reduction of ionization energy for the N1s involved, as counterparts, in the same hydrogen bonding (Figures 3 and 4). The same chemical shifts were found for similar groups by XPS even though in the monomer case they were too closely spaced in energy with other functional groups to be resolved by curve fitting. The greater length of the poly(VGGVG) chains confers flexibility to the chains themselves, that can then rearrange and form a greater number of, and “stronger”, hydrogen bonds. This is confirmed by the FT-IR spectra of poly(VGGVG) and Boc-VGGVG-OEt reported in Figure 7. The amide I zone with two clear signals at 1675 and 1632 cm-1 suggests a well-defined molecular structure for Boc-VGGVG-OEt. By contrast, the more complex amide I structure and the reduced intensity of the band at 1675 cm-1, the H-bond free component, suggests more varied conformations, with several vibrational modes for the hydrogen-bonded, carbonyl-stretching, poly(VGGVG). In the amide II zone, where a contribution from the N-H bending mode overlaps with a minor contribution from the C-N bending mode, no significant differences between the two compounds were observed. Hydrogen bonds are reported to be responsible for the direct interaction between organic polymers and specific surfaces.21-23 Among the possible surfaces, silicon is probably one of the most widely used and was in fact selected in the previous AFM study of poly(VGGVG).6 Here, we have used a Si(100) surface for comparison, characterizing it by XPS before and after the polymer deposition (vide infra) in



Figure 8. C1s region of the Si(100) surface: (a) as received and washed with (b) methanol or (c) distilled water, and left to dry for some minutes. See also caption of Figure 5.

order to verify if the initial event of polymer adsorption was mediated by hydrogen bond formation and, subsequently, also the strength of these bonds. Substrate Characterization. The polymer was suspended in two different media, methanol and distilled water, to understand also the role played by the suspending medium on the supramolecular reorganization. From these suspensions, the polymer was deposited on the Si(100) surface and left to dry for some minutes before introducing it in the analysis chamber. Before deposition, the silicon substrate was characterized by XPS in the “as received” form (a) and after washing it with methanol (b) or distilled water (c). The relevant wide scans (not reported here) showed contributions from silicon and oxygen (oxidized layers) and carbon overlayer.24 The curve fitted C1s regions of these samples are shown in Figure 8: the very low intensity of “extrinsic” background proves that the organic contamination is superficial. The nature of the carbon-containing species is very similar in all cases, even if the total signal intensity is much lower in the methanol case. The fitting parameters for these signals are reported in Table 3. The binding energy of peak A was placed at 285.0 eV and used as a reference to calculate the sample’s surface charging, which resulted in a shift of 1.39, 0.81, and 1.75 eV, for the three cases, respectively. Deposition from Methanolic Suspensions. From previous AFM work,6 poly(VGGVG) is known to follow a timedependent supramolecular evolution when deposited on silicon from methanol suspension: starting as flat platelets, it migrates through a series of ribbonlike structures to a final form of beaded strings. The deposited polymer has been examined by XPS over a period of 5 days, to see if a correspondence could be observed between the supramolecular evolution and the core level’s energy distribution. The resulting four observations, a-d, are shown in Figure 9, whereas the fitting parameters for these spectra are reported in Table 4. In these samples, the carbon region contains signals deriving both from the polymer (peaks 0-4) and substrate

Table 3. Fitting Parameters for the C 1s Spectra Shown in Figure 8

orbital C1s (a)

C1s (b)

C1s (c)

peak no.

BE corr. (eV)

fwhm corrected area % (eV) (arbitrary units) area assignment

A B C D A B C D A B C D

285.0 286.4 287.3 289.0 285.0 286.5 287.4 288.6 285.0 286.6 287.8 289.1

1.75 1.75 1.75 1.75 1.99 1.99 1.99 1.99 1.91 1.91 1.91 1.91

1399.725 207.06 67.10 94.05 460.34 93.41 54.76 52.195 851.16 176.965 70.15 67.13

79.2 11.7 3.8 5.3 69.7 14.1 8.3 7.9 73.0 15.2 6.0 5.8

C (C,H) C-O C)O O-C)O C (C,H) C-O C)O O-C)O C (C,H) C-O C)O O-C)O

contaminants layer (peaks A-D). To separate and assign the energy distribution of peaks 0-4, the contribution from the substrate needed to be subtracted. This subtraction could be satisfactorily performed considering that, as evident also from AFM images,6 the silicon substrate was never fully covered by the deposited polymer. Thus, the relative area ratio of carbon contaminants on silicon and their energy separation were assumed to be the same as that of the pre-deposited substrate after methanol washing. An interesting aspect is that the two sets of peaks have to be “differently” corrected for charging, and moreover, the differential charge changes with time. This variation can be monitored looking at the time-evolving spectra of Figure 9 where the shifting of peak A is evident: In (a) it coincides with peak number 2, revealing that the differential charging between sample and contamination has already moved forward from the beginning of deposition. In spectrum (b), peak A is shifted toward higher binding energies and starts overlapping with peak 3 (but again, as in (a), the curve-fitting could be performed without resolving it as a separate peak), whereas in (c), it appears as a well distinguishable peak at intermediate binding

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Figure 9. Poly(ValGlyGlyValGly) purified, suspended in methanol and deposited on Si(100): C 1s curve fitted regions of (a) just deposited sample; (b) after one night in the analysis chamber (UHV conditions); (c) after one night exposed to the atmosphere; and (d) after 2 days exposed to the atmosphere. See also caption of Figure 5.

energies between peaks 2 and 3. In spectrum (d), the position of peak A is not substantially changed, as compared with spectrum (c). However, in the last case (d), the experimental C:N ratio exceeds the stoichiometric one (see Table 4), most probably because of additional adsorption of surface contaminants during the sample exposure to the atmosphere. The differential charging between polymer and contaminant overlayers of the substrate has certainly made the spectra interpretation more difficult. However, from the analysis of the polypeptide in powder, knowing the positions of peaks 3 and 4, it was possible to determine the number of hydrogen bonds formed for each pentameric unit and to follow their variation with time (Table 4). Also peak 0 was of help, having always the same area ratio against the nitrogen signal. Moreover, the nitrogen signal itself (spectra not shown) appears as a single, almost symmetrical peak, centered at around 400.7 eV, suggesting that the N-H groups are not the H-donors responsible for the splitting of peaks 3 and 4

in the carbon region. Most likely, the polymer is now interacting with the hydroxylated region of the substrate surface,24 through its amide carbonyl groups. Unfortunately, a confirmation from the oxygen region could not be achieved mainly because most of the O1s signal derives from the substrate oxide layer,24 hiding the much weaker one arising from the polymer. A possible picture of the polymer repeating unit and hydroxylated silicon interface is reported in Figure 10: the “interacting” conformation requires approximately half of the carbonyl groups, statistically distributed along the polymer chains, oriented toward the silicon surface and directly interacting through hydrogen bonding. Looking at that picture and considering the concomitant N1s shift toward higher BEs, we could argue that nitrogen also experiences a similar interaction with the hydrated silicon surface, converting to a partially protonated species. Deposition from Water Suspensions. The polymer deposited from water suspensions was also analyzed by


On Elastin-Related Polypeptides Using XPS Table 4. Fitting Parameters for the Carbon 1s Regions of Figure 9, after the Contamination Subtraction (Peaks A-D)a

orbital C1s (a)

C:N C1s (b)

C:N C1s (c)

C:N C1s (d)

C:N a

peak no.

BE corr. (eV)

0 1 2 3 4

283.6 285.0 286.4 288.1 289.4

0 1 2 3 4

283.7 285.0 286.3 287.8 289.1

0 1 2 3 4

283.9 285.0 286.1 287.95 289.25

0 1 2 3 4

284.1 285.0 286.3 288.0 289.3

corrected area normalized fwhm (arbitrary area (eV) units) ratios assignment 1.715 98.75 1.715 570.32 1.715 475.27 1.715 331.525 1.715 157.99 experimental: 1.75 97.71 1.75 575.97 1.75 479.97 1.75 305.57 1.75 280.11 experimental: 1.76 77.27 1.76 465.59 1.76 390.30 1.76 251.53 1.76 213.83 experimental: 1.865 70.92 1.865 452.24 1.865 363.06 1.865 222.26 1.865 204.15 experimental:

1.0 5.9 4.95 3.5 1.65 3.15: 1.0 0.95 5.6 4.7 3.0 2.7 3.17: 1.0 0.9 6.0 5.0 2.6 2.4 3.25: 1.0 0.9 6.2 5.0 2.5 2.3 3.36: 1.0

contam. C (C,H) CR C)O C)O‚‚‚H contam. C (C,H) CR C)O C)O‚‚‚H contam. C (C,H) CR C)O C)O‚‚‚H contam. C (C,H) CR C)O C)O‚‚‚H

The stoichiometric values for the total C:N area ratio are 3.2:1.0.

Figure 10. Schematic and simplified, two-dimensional, representation of the hydrogen bonding for monomeric unit of poly(VGGVG) peptide on the silicon (100) surface.

AFM,6 but in this case, the found supramolecular structures were in the form of stable amyloid-like fibrils not evolving with time toward a different kind of organization. This sample was analyzed by XPS as just deposited on the substrate and after one week. The carbon region spectra are shown in Figure 11, and the fitting parameters for these spectra are in Table 5. The substrate contribution was identified and subtracted as for the methanol sample, this time taking as a reference the substrate as washed with water. As it is clear from both the figure and table, considering the expected influence, on

peak intensities, of additional contaminants after one week’s exposure, the energy distribution is not changing with time and the coincidence of peaks A and 1 binding energies demonstrates that there is no differential charging between sample and contamination. As for the methanol deposition, the oxygen signal deriving from the substrate is too broad to reveal any additional information. The nitrogen signal is again an almost a single peak, centered at 400.3 eV, seemingly slightly skewed toward higher BEs. However, the area ratio of peaks 3 and 4, in the carbon region, is approximately the same as that achieved with time in the methanol suspension (see Tables 4 and 5). Discussion In the previous AFM study,6 synthetic poly(ValGlyGlyValGly) exhibited a differing dynamic behavior according to whether it was deposited from methanol or water suspension onto a silicon substrate. In this work we have repeated the sequences of deposition and encountered a pattern of events that mirror the AFM images. Figure 9 illustrates the changes occurring over a period of time in the C1s spectrum of the polymer deposited from methanol suspension. Peak A is the largest of the peaks associated with the contamination present on the silicon surface prior to deposition and therefore can be better monitored than the associated B-D peaks. These associated peaks move as if experiencing differential charging and, in doing so, change the shape of the entire C1s envelope (Figure 9). Additional spectra, acquired using a range of X-ray fluxes and suspension concentrations, are under study; however, at first sight, it can be said that such a movement is a function of time and not of radiation fluence. It is reasonable to think that it is associated in some way with the changes in molecular coverage of the surface observed by AFM: possible mechanisms would be the lateral diffusion of contaminant molecules to occupy surface sites vacated by adsorbed polymer or the diffusion of contaminant molecules to form some temporary association with the deposited polymer. Notwithstanding the dynamic changes occurring over time, the C1s spectrum obtained from the MeOH deposited material reaches a final stability comparable to that obtained from that deposited from aqueous suspension. Once the “final” C1s XPS spectra are achieved, they show no further change for either form of deposition and it must be assumed that they represent the stable structures: see Figures 9c,d and 11a,b. AFM showed the morphology of the stable structures to be very different, amyloid-like in the case of the aqueous deposition and beaded strings in the case of the methanolic deposition. These two types of final structures appear to be equally stable, especially since the beaded string structure did not convert to an amyloid structure when immersed for a period in water. An indirect indication of structure stabilization, associated with low degrees of freedom and limited vibrational modes, can be deduced by comparing the curve-fitting results reported in the tables throughout this article. In particular, the fwhms for the peaks composing the C1s core level are seen to decrease by going from the monomer to the various polymer forms, reaching

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Flamia et al.

Figure 11. Poly(ValGlyGlyValGly) purified, suspended in distilled water and deposited on Si(100): C1s curve fitted regions of (a) just deposited sample; (b) after one week exposed to the atmosphere. See also caption of Figure 5. Table 5. Fitting Parameters for the Carbon 1s Regions of Figure 11, after the Contamination Subtraction (Peaks A-D)a

peak orbital number C1s (a)

C:N C1s (b)

C:N a

BE corr. (eV)

0 1 2 3 4

283.85 285.0 286.3 287.6 288.8

0 1 2 3 4

283.8 285.0 286.2 287.5 288.8

corrected area normalized fwhm (arbitrary area (eV) units) ratios assignment 1.60 84.05 1.60 524.10 1.60 478.57 1.60 224.73 1.60 173.59 experimental: 3.2: 1.54 49.02 1.54 340.92 1.54 291.43 1.54 178.825 1.54 129.60 experimental: 3.3:

1.0 6.0 5.5 2.6 2.0 1.0 0.8 5.8 5.0 3.0 2.2 1.0

Contam. C (C,H) CR C)O C)O‚‚‚H contam. C (C,H) CR C)O C)O‚‚‚H

The stoichiometric values for the total C:N area ratio are 3.2:1.0.

the narrowest peak widths for the deposited structures (in the methanol case, after taking into account some extra broadening due to differential charging). The additional information obtained with XPS indicates the formation of hydrogen bonds as responsible for such behavior and curve fitting shows a similar number of strong hydrogen bonds in each spectrum. As shown in Figure 10, hydrogen bonds could form between the polymer deposited from methanol and the hydrated silicon substrate. The AFM results show that, in this case, the polymer has a good level of contact with the surface, starting as flat platelets that, showing a Rayleigh instability, migrate in form through a series of ribbonlike structures to a final form of beaded strings. Although this change must involve the making and breaking of the polymer/surface bonds, the polymer has a high degree of contact with the surface at all times. By contrast, the amyloid form deposited from water has, as shown by AFM, little contact with the surface yet forms an equally stable structure with a similar number of strong hydrogen bonds. The nitrogen seems to indicate, in both

cases, its extraneousness as H-donor in the formed hydrogen bonds even if contributing somehow to the structures stability as H-acceptor. On the other hand, when the polymer was examined on its own (see powdered polymer) both IR and XPS coherently indicate the presence of hydrogen bonds involving >CO and >NH groups, therefore suggesting the presence of classical, intermolecular β-structures. One can speculate that the surface bonds that “lock in” the structure formed from methanolic deposits are replaced by hydrogen bonds in the aqueous deposits, perhaps involving water molecules. Unfortunately, XPS can only monitor water by means of its oxygen that was, as already said, fully masked by the superimposing oxygen from the hydrated silicon substrate, and therefore, this speculation cannot be confirmed. What is very important is that both structures were demonstrated to appear in elastin, even if under quite different conditions. As a matter of fact, the beaded-string like filaments represent the characteristic supramolecular organization of elastin, whereas amyloid-like fibrils have been very recently evidenced for the polypeptide sequence coded by exon 30 of human tropoelastin.25 In the latter case, a hypothesis has been put forward linking the deposition of the amyloid fibers to some diseases such as acute interstitial lung pathology and deposition of elastotic material in arteries. Therefore, it is clear that poly(ValGlyGlyValGly) constitutes a simple, yet excellent, model for the molecular and supramolecular properties of elastin. In addition, the results obtained in the present paper allow further insight into the molecular features leading to the supramolecular organization. In particular (1) no interchain hydrogen bonds seem to be present in the organization of the classical beaded-strings (as seen, e.g., by electron microscopy1) when deposited on a substrate; (2) in the case of amyloid fibers, where our results exclude a significant attachment to the substrate, the only possible interpretation is that proposed in Figure 12. Accordingly, an antiparallel β-structure, comprising watermediated interchains hydrogen bonds, seems to account for



stabilizing the amyloid structure, is mediated by the strong polarity of the water suspending medium. Acknowledgment. The sample transfer and XPS acquisition were carried out with the technical assistance of Dr. F. Langerame. R.F. thanks Dr. Neluta Ibris for his constant help during experiments. A.M.T. acknowledges the European Community (Grant QLK6-2001-00332) and MIUR (Grant COFIN 2002). References and Notes

Figure 12. Schematic and simplified representation of water-bridged hydrogen bonding between monomeric units of poly(VGGVG) peptide in amyloid fibers.

a novel amyloid structure whose details deserve to be deeply investigated in the next future. More generally, future perspectives of this study include a combined use of XPS and AFM together with other surface techniques to be used in parallel. The behavior of other synthetic polypeptidic sequences of elastin will be also examined in relation to different substrate surfaces and suspending solvents. Conclusion The study of synthetic sequences of elastin is important because they mimic the supramolecular assembly of the elastin itself.1-6,20 The component peaks of the XPS spectra have been assigned and are in agreement with the area ratios predicted from the molecular formula augmented by molecular modeling for the pentapeptide monomer. Deposition from water produces a stable XPS spectrum but in the case of deposition from methanol a spectrum of similar stability forms over the course of time. In both cases, peak splitting for >CO groups due to the formation of strong hydrogen bonds has been observed, although, at this stage, the partner to these bonds has not been identified because of the overwhelming presence of oxidized silicon from the substrate. As modeled in Figures 10 and 12, we have proposed that the formation of a beaded string structure is mediated by the formation of hydrogen bonds to the silicon surface whereas the formation of intermolecular hydrogen bonds,

(1) Debelle, L.; Tamburro, A. M. Int. J. Biochem. Cell Biol. 1999, 31, 261. (2) Martino, M.; Tamburro, A. M. Biopolymers 2001, 59, 29. (3) Martino, M.; Perri, T.; Tamburro, A. M. Biomacromolecules, 2002, 3, 297. (4) Martino, M.; Perri, T.; Tamburro, A. M. Macromol. Biosci. 2002, 2, 319. (5) Martino, M.; Coviello, A.; Tamburro, A. M. Int. J. Biol. Macromol. 2000, 27, 59. (6) Flamia, R.; Zhdan, P. A.; Martino, M.; Castle, J. E.; Tamburro, A. M. Biomacromolecules 2004, 5, 1511. (7) Briggs, D.; Seah, M. P. Practical Surface Analysis; Wiley: Chichester, U.K., 1990; Vol. 1. (8) Beamson, G. J. Electron Spectrosc. Relat. Phenom. 2001, 121, 163. (9) Aplincourt, P.; Bureau, C.; Anthoine, J.-L.; Chong, D. P. J. Phys. Chem. A 2001, 105, 7364. (10) Castle, J. E.; Salvi, A. M. J. Electron Spectrosc. Relat. Phenom. 2001, 114-116, 1103 and references therein cited. (11) Castle, J. E.; Chapman-Kpodo, H.; Proctor, A.; Salvi, A. M. J. Electron Spectrosc. Relat. Phenom. 1999, 106, 65. (12) Shirley, D. A. Phys. ReV. B 1972, 5, 4709. (13) See ref 7: Appendix 6. (14) Wagner, C. D.; Davis, L. E.; Zeller, M. V.; Taylor, J. A.; Raymond, R. M.; Gale, L. H. Surf. Interface Anal. 1981, 3, 211. (15) See ref 7: Seah, M. P. Chapter 5. (16) Seah, M. P. Surf. Interface Anal. 1986, 9, 85. (17) Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Elbert, S. T.; Gordon, M. S.; Jensen, J. H.; Koseki, S.; Matsunaga, N.; Nguyen, K. A.; Su, S. J.; Windus, T. L.; Dupuis, M.; Montgomery, J. A. J. Comput. Chem. 1993, 14, 1347. (18) Rossi, A. R.; Sanda, P. N.; Silverman, B. D.; Ho, P. S. Organometallics 1987, 6, 580. (19) Lanza, G. et al. manuscript in preparation. (20) Guantieri, V.; Grando, S.; Pandolfo, L.; Tamburro, A. M. Biopolymers 1990, 29, 845. (21) Xiao, S. J.; Textor, M.; Spencer, N. D.; Wieland, M.; Keller, B.; Sigrist H. J. Mater. Sci.: Mater. Med. 1997, 8, 867. (22) Zhang, F.; Kang, E. T.; Neoh, K. G.; Wang, P.; Tang, K. L. J. Biomed. Mater. Res. 2001, 56, 325. (23) Giannoulis, Constantina, S.; Tejal, A. J. of Mater. Sci.: Mater. Med. 2002, 13, 75. (24) McCafferty, E.; Wightman, J. P. Surf. Interface Anal. 1998, 26, 549. (25) Tamburro, A. M.; Pepe, A.; Bochicchio, B.; Quaglino, D.; Ronchetti, I. P. J. Biol. Chem. 2005, 280, 2682.

BM049290S

Conformational Study and Hydrogen Bonds Detection on Elastin

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