ARTICLE pubs.acs.org/JPCC
Probing the Binding Mechanism of Peptides on a Copper Surface: Multilayer Self-Assembly Promoted by Glutamate Residues C. Methivier, V. Lebec, J. Landoulsi,* and C.-M. Pradier Laboratoire de Reactivite de Surface, UMR CNRS 7197, Universite Pierre et Marie Curie-Paris VI, Site d’Ivry-Le Raphael, 94200 Ivry-sur-Seine, France ABSTRACT: Short peptides interact strongly with copper surfaces. We report here a full characterization on the interaction of Gly-Pro and Gly-Pro-Glu with the Cu(110) surface. By combining infrared analysis with X-ray photoelectron spectroscopy, two binding centers have been identified: the carboxylate and the amino groups. Moreover, spectral data were used in an innovative way to evaluate the number of nitrogen atoms per molecule bound to the surface. The presence of additional glutamate fragment in the peptide modifies the adsorption mechanism radically, leading to multilayer self-assembling growth. Our findings open up new insights into interfacial phenomena involved in intermolecular bonds and molecule-surface interactions.
1. INTRODUCTION Molecular recognition involving proteins, nucleic acids, or aptamers governs all biological structures and functionnalities, such as hard tissue biomineralization, transport and storage molecules, immune protection, and cell differentiation.1 In particular, specific interactions with inorganic materials have opened routes to design new hybrid materials with versatile functions.2 Recent progress in combinatorial biology has permitted the identification of peptides that are able to recognize specifically inorganic materials.3,4 The specificity of these binders is now exploited in various applications, such as self-assembly for surface biocompatibility,5,6 drug delivery,7 and crystal growth regulation.8,9 However, little is known about the mechanism(s) by which specific amino acid sequences interface coherently with specific solid surfaces. Two parameters are of major importance: (i) the site of the specific recognition by the peptide and (ii) the role of the supramolecular organization of peptides at solid surfaces. In addition to common global approaches broadly used to characterize biointerfaces at the macro- or microscopic scale, a molecular approach based on the use of small biomolecules has led to great advances in understanding interfacial mechanisms.10,11 In this context, we have recently shown that short peptides may be adsorbed under controlled ultrahigh vacuum (UHV) conditions to investigate their interactions with solid surfaces.12-14 Influences of the molecule size, the amino acid sequence or the adsorption conditions (evaporation time, temperature) were investigated. Furthermore, relevant information about peptide-inorganic surface interaction, including the binding mechanism, conformational changes and structural stability, have been brought up using molecular dynamic simulations15-18 or density functional theory.19,20 Accordingly, experiments performed under model conditions may be reinforced by theoretical studies in a reasonable way to better understand the mechanisms involved in the peptide/surface interface at the molecular level. r 2011 American Chemical Society
Although adsorption of amino acids on inorganic surfaces has been broadly investigated under UHV conditions,11 adsorbing short peptides is still rare. To our knowledge, in addition to studies reported by our group, only a few papers have examined the adsorption of peptides under UHV conditions.19-24 In previous papers, we reported successful adsorption of peptides containing two or three amino acids onto gold surfaces.14 In the present study, we focus on the adsorption mechanism of GlyPro-Glu peptide onto a Cu(110) surface. This sequence is an important N-terminal peptide of the insulin-like growth factor and is involved in numerous biological processes.25 Copper was chosen as a biologically relevant surface known to complex a wide range of peptides in aqueous media.26 Furthermore, it has been broadly used to investigate the adsorption of amino acids.27-32 In this study, the adsorption behavior of Gly-Pro-Glu tripeptide is compared with that of Gly-Pro dipeptide with a view to better understanding the role of additional carboxylic groups originating from glutamate, possibly involved in both surface-molecule and molecule-molecule interactions. Particular attention is given to the chemical state of adsorbed molecules and the nature of binding groups to the copper surface. To this end, in situ infrared characterization was combined with XPS analysis performed immediately after various exposure times.
2. EXPERIMENTAL SECTION 2.1. Materials. Gly-Pro and Gly-Pro-Glu peptides (molecules depicted in Figure 1), were purchased from Bachem (99%) and used without further purification. It was contained in a small electrically heated glass tube, separated from the main vacuum chamber by a gate valve, and differentially pumped by a turbomolecular pump. Before Received: September 26, 2010 Revised: January 18, 2011 Published: February 23, 2011 4041
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Figure 1. Schematic structures of (a) Gly-Pro and (b) Gly-Pro-Glu peptide molecules.
sublimation, peptides were outgassed for at least 1 h at 370 K with the gate valve closed. Peptides were then heated to 400 K and exposed to the copper crystal. During sublimation, the main chamber pressure typically rose to 1 10-8 Torr. Experiments were carried out in multitechnique UHV chambers, facilitating polarization modulation reflection absorption infrared spectroscopy (PM-RAIRS), Auger electron spectroscopy (AES), and low-energy electron diffraction (LEED). The Cu(110) single crystal was routinely cleaned in vacuum (base pressure equal to 1 10-10 Torr) by cycles of Arþ ion sputtering (PAr = 2 10-6 Torr, 250 V) and annealing to 850 K. The surface cleanliness and structure were checked by AES and LEED, respectively, prior to peptide adsorption. 2.2. In Situ PM-RAIRS Measurements. Spectra were recorded using a Nicolet 5700 spectrometer equipped with a nitrogen-cooled MCT wide-band detector. A ZnS grid polarizer and ZnSe photoelastic modulator to modulate the incident beam between p and s polarization were placed prior to the sample. The spectrometer was interfaced to the UHV chamber via ZnSe windows. The reflected light was focused onto the detector at an optimal incident angle of 85°. All spectra were obtained after 512 scans at 8 cm-1 resolution. 2.3. XPS Analyses. For XPS analyses, the sample was transferred to the adjacent UHV XPS chamber immediately after peptide evaporation at the desired exposure time. Analyses were performed using a SPECS (Phoibos MCD 150) spectrometer (SPECS, Germany) equipped with a nonmonochromatized aluminum X-ray source (hν = 1486.6 eV) powered at 10 mA and 15 kV, and a Phoibos 150 hemispherical energy analyzer. The resulting analyzed area was 5 mm diameter. A pass energy of 20 eV was used for survey scan and 10 eV for narrow scans. The single crystal was fixed on the support, and no charge stabilization device was used on this conducting sample. The pressure in the analysis chamber during measurement was around 10-10 Torr. The photoelectron collection angle, θ, between the normal to the sample surface and the analyzer axis was 0°. The following sequence of spectra was recorded: survey spectrum, Cu 2p, O 1s, N 1s, C 1s, and CuLMM. The binding energy scale was set by adjusting the Fermi level to zero. The data treatment was performed with the Casa XPS software (Casa Software Ltd., UK). The peaks were decomposed using a linear baseline, and a component shape defined by the product of a Gauss and Lorentz function, in a 70:30 ratio. Molar concentration ratios were calculated using peak areas normalized according to Scofield factors.
3. RESULTS AND DISCUSSION 3.1. Chemical State of Peptides. One major feature to examine the binding mechanism is the identification of chemical groups present in the peptide molecules when adsorbed on the surface. XPS analyses were used to this end. As both Gly-Pro and Gly-Pro-Glu molecules were sublimated in the UHV chamber, XPS analyses on the peptide powder were recorded prior to the
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adsorption on the copper surface. Typical C 1s, O 1s, and N 1s XPS peaks are presented in Figure 2. The C ls peak was decomposed in four components, the fwhm of which were imposed to be equal: a component at 285.0 eV due to carbon only bound to carbon or hydrogen [C-(C, H)]; a component at about 286.3 eV due to carbon making a single bond with oxygen or nitrogen [C-(O, N)]; a component at 288.0 eV due to carbon making one double bond with oxygen [CdO] in amide or carboxylate; and the last component near 289.0 eV attributed to carbon in carboxyl function [(CdO)-OH].33 The O 1s peak was decomposed into two components with the same fwhm. The first one at ∼531.6 eV was attributed to oxygen making a double bond with carbon (CdO including amide and carboxyl) and to oxygen of carboxylate.33 The second component, at 533.3 eV, can be attributed to oxygen making single bonds with carbon (C-OH of carboxyl).33 The N 1s peak showed a main component at 400.3 eV attributed to amide or amine (N-C). An additional component appeared clearly near 401.8 eV, indicating the presence of protonated amines (Figure 2). Molar concentrations associated with the components of C 1s, O 1s, and N 1s peaks, ratioed to total carbon, are given in Table 1. XPS spectra recorded on the copper surface after peptide evaporation are given in Figure 3. The major differences, compared with peptide powders, appeared in the N 1s region, showing a component at ∼398.6 eV in addition to one main contribution at 400.3 eV. The assignment and the chemical nature of this low binding energy component will be discussed in Section 3.2. Note that the absence of contribution near 401.8 eV, unlike the peptide powder (Figure 2), indicates the absence of a significant amount of protonated amines. The O 1s and C 1s peaks recorded on the adsorbed peptides revealed a decrease of the contribution at 533.3 and 289.0 eV, respectively. These two components totally disappeared in the case of the GlyPro dipeptide, suggesting the disappearance of carboxyl acid functions (COOH) to the benefit of carboxylates (COO-) (Figure 3). These results evidence that the dipeptide tends to adsorb in the anionic state on the copper surface. Conversely, the presence of COOH groups in the tripeptide when adsorbed on the copper surface is evidenced by XPS components at 289.0 and 533.3 eV in the C 1s and O 1s peaks, respectively (Figure 3b). However, the molar ratios related to the amount of carboxyl groups in the adsorbed molecule [(CdO)-OH/C], obtained from XPS, is appreciably lower than the theoretical value computed from the molecule stoichiometry (Table 1). This suggests that part of the COOH groups is, indeed, deprotonated. By combining these observations and those provided by the N 1s peak (absence of NH3þ groups, Figure 3b), it is reasonable to conclude that the tripeptide adsorbs mainly in the anionic state and that the molecule includes a small amount of COOH groups (about 20%, data from Table 1). A full comparison between the expected atomic ratios for both peptides and those measured by XPS from powder or from adsorbed peptides is given in Table 1. Interestingly, good agreement can be noticed, suggesting that both tri- and dipeptides are still intact when adsorbed onto the copper surface, and no other species are adsorbed. 3.2. Binding Mechanism of Peptides on Copper Surface. The interactions between the copper surface and the peptides were then investigated as a function of the exposure time. To this end, the adsorption of Gly-Pro and Gly-Pro-Glu on the copper surface was monitored in situ using infrared analyses. Figure 4 4042
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Figure 2. O 1s, N 1s, and C 1s XPS peaks recorded on (a) Gly-Pro and (b) Gly-Pro-Glu powder used for evaporation under UHV conditions.
Table 1. Mole Concentration Ratios with Respect to Total Carbon (a) Computed Using the Theoretical Stoichiometry of Peptides or Determined from XPS Peak Component As Assigned, Recorded on (b) the Peptide Powder, (c) Adsorbed on the Copper Surface C 1s (eV)
O 1s
285.0
286.3
288.0
289.0
531.6
533.1
C-(C, H)
C-(O, N)
CdO
(CdO)-OH
CdO
C-OH
N 1s 398.6
400.3
Otot
N398.6
C-N
Ntot
(a)
0.29
0.43
0.14
0.14
0.29
0.14
0.43
-
0.29
0.29
(b)
0.28
0.43
0.24
0.06
0.35
0.04
0.39
-
0.25
0.25
(c)
0.32
0.47
0.21
0.00
0.23
0.00
0.23
0.08
0.15
0.23
Gly-Pro-
(a)
0.33
0.33
0.17
0.17
0.33
0.17
0.50
-
0.29
0.25
Glu
(b)
0.34
0.34
0.24
0.08
0.36
0.08
0.44
-
0.22
0.22
(c)
0.34
0.34
0.26
0.07
0.34
0.03
0.37
0.04
0.12
0.15
Gly-Pro
shows the evolution of the PM-RAIRS spectra as a function of the evaporation time of both peptides on the Cu(110) surface. After 1 min of exposure, the spectrum obtained with Gly-Pro exhibits only one absorption band at 1604 cm-1 due to the COOasymmetric stretching (Figure 4a). With increasing exposure time, this band is kept almost unchanged in terms of position and intensity, whereas weak bands emerging from the broad massif at 1423 and 1458 cm-1 can be observed, attributed to ν(COO-) and δ(CH2), respectively (Figure 4a). Results obtained on GlyPro-Glu are appreciably different (Figure 4b). At low exposure, spectra show the presence of both the asymmetric and symmetric COO- stretch at 1623 and 1415 cm-1, respectively. Moreover, other easily identifiable peaks include C-H deformation around 1435 cm-1 and CdO stretch in the COOH group at 1716 cm-1. The latter band splits into two contributions at 1670 and 1739 cm-1 after 15 and 60 min, indicating the presence of two types of acids groups. The band at 1674 cm-1 is typical of H-bonded cyclic acid dimers, leading to an appreciable frequency downshift,
and the one at 1739 cm-1 is due to open chain acid groups involving less H-bonding.34 These findings suggest that peptide molecules interact with the surface via carboxylate (COO-) and with each other through H-bonding between carboxyl acid (COOH) groups. These results, in agreement with XPS data detailed above, point out the role of the COO- functions in the molecule-surface interactions. LEED pattern was not observed after the adsorption of di- or tripeptides, independently of the exposure time, suggesting that no 2D organization is formed, in contrast to what had been observed for the same peptides on the Au(110) surface at low exposure time.14 Regarding the evolution of the chemical state of the adsorbed peptides as a function of the evaporation time, discrimination between NH2 and NH3þ groups is needed. This is not easy from vibrational data, but the absence of a band around 15001550 cm-1 suggests that the adsorbed di- and tripeptides did not contain protonated amino groups35 and that they adsorb onto 4043
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Figure 3. O 1s, N 1s, and C 1s XPS peaks recorded on the Cu(110) surface after evaporation of (a) Gly-Pro and (b) Gly-Pro-Glu under UHV conditions.
Figure 4. PM-RAIRS spectra recorded on Cu(110) as a function of the evaporation time of (a) Gly-Pro and (b) Gly-Pro-Glu peptides under UHV conditions.
the copper surface in the anionic state, similarly to most amino acids on copper.11 The use of XPS analysis provides relevant information on the protonation level of amino groups, as discussed above. Indeed, in the N 1s region, the discrimination between the components due to primary (∼400.3 eV) and to protonated amines (∼401.8 eV) can be unambiguously made like in polymers36 or in other biosystems.33 The N 1s peak recorded after the adsorption of the dipeptide on the copper surface did not change with increasing exposure time (Figure 5a). It shows two components at about 400.3 and 398.6 eV, but no contribution due to protonated amine was observed. The tripeptide exhibited the same behavior at low exposure time (Figure 5b); however, with the exposure time, the
Figure 5. N 1s XPS peaks recorded on Cu(110) as a function of the evaporation time of (a) Gly-Pro and (b) Gly-Pro-Glu peptides under UHV conditions.
contribution due to primary amines and peptidic links (∼400.3 eV) increased markedly with respect to the one at lower binding energies (∼398.6 eV). Furthermore, the presence of a contribution due to protonated amine can be observed after 60 min of exposure (Figure 5b). Interestingly, in the N 1s region, the contribution at low binding energy (∼398.6 eV) is clearly visible independently of the peptide and the exposure time, except after 60 min of GlyPro-Glu adsorption (Figure 5b). This contribution, previously assigned to imine nitrogen (dN-),37,38 cannot be attributed to 4044
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nitrogen originating from the proline fragment, in this study, since it was not observed in XPS analysis of the Gly-Pro powder (Figure 2a). Furthermore, the adsorption of proline amino acid on other substrates did not reveal such a contribution.39,40 The appearance of this contribution in the N 1s region may be linked to the bond established between the nitrogen of the peptide and the copper surface through a hydrogen atom (N-H 3 3 3 Cu), in agreement with a recent study.20 The involvement of -NH2 groups in the adsorption mechanism of amino acids is, indeed, reported, but chemical evidence was not clear. Actually, amino acids and peptides are known to bind to metal surfaces involving a mechanism analogous to that observed upon the formation of organometallic complexes in solutions.21 Previous studies regarding reactions of metal ions in peptide solutions indicate that the most common site for coordination is the terminal amino group (-NH2).26 Following this interpretation, in the present study, one innovative way to probe molecular binding of the adsorbed peptides on a copper surface consists in estimating the fraction of Cu-bound nitrogen atoms per molecule. This can be made since the contribution due to nitrogen bound to the copper surface (at ∼398.6 eV) and the one due to the remaining nitrogen in the molecule (at ∼400.3 eV) are easily separated (Figure 5). On the basis of XPS data, the number n of nitrogen atoms per peptide molecule bound to copper can be computed using the following equation: n ¼ a
N398:6 Ntot
ð1Þ
where a is the number of nitrogen atoms in the peptide molecule (a = 2 in Gly-Pro and 3 in Gly-Pro-Glu, Figure 1). N398.6 is the molar concentration associated with the component at 398.6 eV in the N 1s peak and Ntot is the total concentration of nitrogen (data from Table 1). It is worth noting that this parameter (n) has a physical meaning only at low exposure time; that is, at early stage when the substrate has a dominating influence on the adsorption mechanism. Thus, molecule-molecule interactions are negligible with respect to molecule-substrate interactions. Unexpectedly, no significant differences can be noticed between the di- and tripeptide: the n values obtained are 0.76 and 0.78 for Gly-Pro and Gly-Pro-Glu, respectively, suggesting the following comments. On one hand, the presence of glutamate in the tripeptide molecule did not imply additional nitrogen-copper binding site, because both peptides exhibit the same trend. On the other hand, the value obtained, n ∼0.8, suggests that less than one nitrogen atom per molecule is involved in the peptide-surface binding. Indeed, proline does not contain an -NH2 group, but rather, the secondary dNH group. Hence, when inserted in a peptide chain, deprotonation may not occur so that the nitrogen atom is unable to bond to copper.41 Moreover, the involvement of protonated nitrogen of the amide bond, in the Gly-Pro-Glu peptide, in a link with the metal can be excluded because it would imply an energetically unfavorable configuration.42 Thus, in addition to COO- functions, both peptides strongly interact with the copper surface via nitrogen of the amino group. 3.3. Monolayer vs Multilayer Adsorption. It was clearly shown for Gly-Pro-Glu on copper that the contribution due to primary amines and peptidic links in the N 1s peak (at∼400.3 eV) increased markedly with respect to the one at lower binding energies (∼398.6 eV), when increasing the exposure time (Figure 5b). This indicates that the amount of adsorbed tripeptides increases with
Figure 6. Evolution of the adsorbed layer thickness as a function of the evaporation time of (O) Gly-Pro or (b) Gly-Pro-Glu.
Figure 7. Schematic representation of adsorption mode of (a) Gly-Pro, (b) Gly-Pro-Glu on a Cu(110) surface.
exposure time, in contrast to that of dipeptide, which reaches a saturation. The evaluation of the average adsorbed layer thickness, d, at various exposure times, on the basis of XPS results, provides more convincing data. This can be computed using the following equation: " !# -d ad ad λC CC 1 - exp ad λC cos θ ½C iCu σC ! ¼ ð2Þ ½Cu iC σ Cu -d su su λCu CCu exp ad λCu cos θ Cos θ is 1 because the photoelectron collection angle θ is equal to zero. iC and iCu are the relative sensitivity factors of C and Cu, respectively, provided by the spectrometer manufacturer. The Scofield photoionization cross sections σ are 1 for C 1s and 2.48 for Cu 2p. The superscripts ad and su designate the adsorbed layer and the copper substrate, respectively. Cyx are concentrations of the element x in the matrix y. The electron inelastic mean free paths were calculated using the Quases program based on the TPP2M formula.43 The average thickness of the Gly-Pro-Glu layer markedly increased with the exposure time, suggesting a multilayer growth regime (Figure 6). In contrast, the Gly-Pro adsorbed layer rapidly reached a saturation value around 0.6 nm, consistent with one peptide monolayer (Figure 6). All these findings lead to the adsorption mechanism depicted in Figure 7. Both peptides adsorb on the copper surface in the anionic state via (i) COO- groups, probably involving electrostatic attractions with slightly positively charged copper atoms; and (ii) the nitrogen of the NH2 group, originating from the glycine fragment, thus leading to the formation of a relatively strong binding with copper. In contrast to Gly-Pro, the tripeptide is able to assemble in multilayers, thanks to the carboxylic groups of glutamate residues which make H-bond intermolecular connections (Figure 7), as revealed by infrared and XPS analyses. The presence of COOH is supported by the presence of contributions at 289.0 and 533.3 eV in the XPS C 1s and O 1s peak, respectively, solely for the tripeptide, as 4045
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4. CONCLUSIONS The adsorption of Gly-Pro and Gly-Pro-Glu peptides on a copper surface was performed under UHV conditions. A full characterization based on PM-RAIRS and XPS analyses provided relevant information regarding the surface-molecule and molecule-molecule interactions involved in the adsorption mechanism. To summarize, both Gly-Pro and Gly-Pro-Glu adsorb and interact with the copper surface via their carboxylate groups and the nitrogen atom of their amino group. The major role played by the glutamate fragment in the tripeptide consists in enhancing intermolecular interactions through H-bonded carboxyl groups and, thus, promoting the formation of multilayers. ’ AUTHOR INFORMATION Corresponding Author
*Phone: þ33 (0)1 44 27 23 98. Fax: þ33 (0)1 44 27 60 33. E-mail:
[email protected].
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