Structure of Monolayers Formed from Neurotensin and Its Single

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Structure of Monolayers Formed from Neurotensin and Its Single-Site Mutants: Vibrational Spectroscopic Studies Edyta Podstawka-Proniewicz,*,† Andrzej Kudelski,‡ Younkyoo Kim,§ and Leonard M. Proniewicz†,^ †

Faculty of Chemistry, Jagiellonian University, ul. Ingardena 3, 30-060 Krakow, Poland Faculty of Chemistry, University of Warsaw, ul. L. Pasteura 1, 02-093 Warsaw, Poland § Department of Chemistry, Hankuk University of Foreign Studies, Yongin, Kyunggi-Do, 449-791, Korea ^ The State Higher Vocational School, ul. Mickiewicza 8, 33-100 Tarnow, Poland ‡

bS Supporting Information ABSTRACT: The human, pig, and frog neurotensins and four single-site mutants of human neurotensin (NT), having the following modifications, [Gln4]NT, [Trp11]NT, [D-Trp11]NT, and [D-Tyr11]NT, were immobilized onto an electrochemically roughened silver electrode surface in an aqueous solution. The orientation of adsorbed molecules was determined from surfaceenhanced Raman scattering (SERS) measurements. A comparison was made between these structures to determine how the change upon the mutation of the neurotensin structure influences its adsorption properties. The SERS patterns were correlated with the contribution of the structural components of the aforementioned peptides to the ability to interact with the NTR1 G-protein receptor. Briefly, the SERS spectra revealed that the substitution of native amino acids in investigated peptides influenced slightly their adsorption state on an electrochemically roughened silver surface. Thus, human, pig, and frog neurotensins and [Gln4]NT and [D-Tyr11]NT tended to adsorb to the surface via the tyrosine ring, the oxygen atom of the deprotonated phenol group of Tyr11, and the CH2 unit(s), most probably of Tyr11, Arg9, and/or Leu13. The observed changes in the enhancement of the deprotonated Tyr residue SERS signals indicated a further parallel orientation of a phenolO bond with regard to the silver surface normal for pig NT, [Gln4]NT, and [D-Tyr11]NT, whereas the orientation was slightly tilted for human and frog NT. In the case of [Trp11]NT and [D-Trp11]NT, the formation of a peptide/Ag complex was confirmed by strong SERS bands involving the phenyl co-ring of Trp11/D-Trp11 and CH2 vibrations and the tilted and flat orientations of the two compounds with respect to the surface substrate. The spectral features were accompanied by a SERS signal caused by vibrations of the carboxyl group of C-terminal Leu13 and the guanidine group of Arg9. Reported changes in SERS spectra of L and D isomers were fully supported by generalized two-dimensional correlation analysis. Additionally, a combination of mutation-labeling and vibrational spectroscopy (Fourier-transform Raman and absorption infrared) was used to investigate the possible peptide conformations and environments of the tyrosine residues.

’ INTRODUCTION An androgen-sensitive prostate cancer cell line, LNCaP, produces and secretes a G-protein-linked neuropeptide, called neurotensin, during androgen deprivation as an effect of both increased neuropeptide synthesis/secretion and decreased neuropeptide-directed proteolytic activity. This leads to the development of a positive growth-regulatory loop involving neurotensin.1 Neurotensin (NT, pGlu1-Leu2-Tyr3-Glu4-Asn5Lys6-Pro7-Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13OH, where pGlu denotes 5-oxo-proline; all amino acids exist in the L isomer) is an endogenous tridecapeptide originally isolated in 1973 by Carraway and Leeman from bovine hypothalami.2 It is distributed heterogeneously both in the central nervous system and in the gastrointestinal tract,3,4 exerting a broad spectrum of biological functions, such as the regulation of dopamine transmission,5 anterior pituitary hormone secretion,6 reduction of locomotor activity, barbiturate sedation, hypothermia,7 muscle relaxation,8 potentiation of the glucose level in plasma,9 r 2011 American Chemical Society

analgesia,10 and proliferation of normal11 and neoplastic cells.12,13 This last effect has been of particular interest because it could strengthen our understanding of the impact of NT on the stimulation, on a molar basis (10 and 100 pM),14 of the growth of prostate cancer.15 For this reason, NT agonists are useful from a therapeutic point of view. Unfortunately, native NT has not been a suitable in vivo carrier for delivering imaging agents to neurotensin-specific receptors present in tumor tissues because of its short half-life (1.5 min) in human plasma.16 This limitation could be overcome by a simple stabilization strategy, which would replace the metabolically active amino acid in the peptide sequence with mimics (mutations) that would stabilize the entire molecule and retain a high NT binding affinity.17,18 Received: January 25, 2011 Revised: April 20, 2011 Published: May 04, 2011 6709

dx.doi.org/10.1021/jp200805f | J. Phys. Chem. B 2011, 115, 6709–6721

The Journal of Physical Chemistry B

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Table 1. Amino Acid Sequences of Human, Pig, and Frog Neurotensin and Single-Site Mutants of Human Neurotensin, [Gln4]NT, [Trp11]NT, [D-Trp11]NT, and [D-Tyr11]NT amino acid sequence 1

2

3

4

5

6

7

8a

9a

10a

11

12a

13a

NT

pGlu

Leu

Tyr

Glu

Asn

Lys

Pro

Arg

Arg

Pro

Tyr

Ile

LeuOH

pig NT

pGlu

Leu

Tyr

Glu

Asn

Lys

Ser

Arg

Arg

Pro

Tyr

Ile

LeuOH

frog NT [Gln4]NT

pGlu pGlu

Ser Leu

His Tyr

Ile Gln

Ser Asn

Lys Lys

Ala Pro

Arg Arg

Arg Arg

Pro Pro

Tyr Tyr

Ile Ile

LeuOH LeuOH

[Trp11]NT

pGlu

Leu

Tyr

Glu

Asn

Lys

Pro

Arg

Arg

Pro

Trp

Ile

LeuOH

[D-Trp11]NT

pGlu

Leu

Tyr

Glu

Asn

Lys

Pro

Arg

Arg

Pro

D-Trp

Ile

LeuOH

[D-Tyr11]NT

pGlu

Leu

Tyr

Glu

Asn

Lys

Pro

Arg

Arg

Pro

D-Tyr

Ile

LeuOH

peptide

a

a

a

Common amino acid in the sequence of all the investigated peptides.

To date, there have been three known subtype receptors belonging to either the superfamily of G-protein-coupled receptors (NTR1 and NTR2), sharing 43% amino acid sequence identity and 64% structural identity,1921 or the family of sorting receptors (NTR3),22 to which NT binds with different affinities. Various experiments using NT receptor antagonists have indicated that most of the activity of NT results from its interaction with NTR1.2325 However, the mode of these interactions remains unknown. Results obtained with partial sequences of NT have demonstrated that binding occurs through so-called “receptor” sites, which involve the C-terminal region of NT and are analogous to those involved in the pharmacological and/ or biological effects of this peptide. Briefly, the C-terminal hexapeptide (namely, NT813) possesses all of the sequence requirements for full binding potency to NTR1.26,27 When the C-terminal fragment is reduced to five residues, as in the NT913 pentapeptide, a dramatic decrease in binding potency is observed, indicating that Arg8 has an essential role in binding, as anticipated from pharmacological in vivo studies.28 The free Leu13COOH terminus of NT is important for reactivity with NTR1, as evidenced by the almost complete loss of binding potency observed with the NT112 dodecapeptide. Blockage of the free carboxyl group demonstrates that a charged group is required at that position.29 In addition, a retro-inverso modification of the charged group (substitution by D-Leu), or of residues adjacent to it, results in a decrease of the ionic interactions involved at the binding site.29 Additionally, the Tyr residue at position 11 of the amino acid sequence (Tyr11) has been shown to play a crucial role in the NT interaction with NTR1.27,31,32 Therefore, it is likely that an incorporation of a large spherical amino acid on the phenol side chain of Tyr11 (i.e., Arg9 or Leu13) would alter the biological activity of NT. However, the removal of a hydroxyl group, by replacing Tyr11 (or Tyr3) with L-phenylalanine, yields a peptide with a binding potency equivalent to that of neurotensin.29 Modifications in either arginine residue, Arg10 or Arg9, produce a few-fold increase in the binding of mutated neurotensin relative to the parent molecule. Additionally, any changes from residues Pro7 through the C-terminal Leu13 considerably reduce the NT receptor affinity. Several reports have also suggested that cleavage of the Pro10Tyr11 bond is a key event in the physiological process that leads to neurotensin inactivation. Thus, several authors have described the enhanced potency in vivo of neurotensin analogues in which Tyr11 is substituted by a D-amino acid.3033 Furthermore, the extent of alternation in NT binding properties inferred with the presence of neurotensin mutations indicates that the amidation

of Glu4 (Glu4 f Gln4) does not enhance or reduce the biological activities of NT.34 Additionally, replacement of the metabolically active L-amino acid by its Disomer within the NH-terminal portion of neurotensin, residues pGlu1 through Pro7, exerts essentially no effect on the binding of neurotensin.29 Despite the broad range of biological and clinical importance of neurotensin, very little is known regarding its structure or the manner of its interaction with NTR1. Progress in this area could provide some insight into the neurotransmiter’s function and increase our understanding of the specificity of its binding to the receptor. Such information is crucial to designing novel bioactive peptides with great putative pharmacological interest, that is, antagonists or carrier molecules for targeting cytotoxic drugs to tumor cells. The locations of the modifications that give rise to antagonists are usually determined by the location of the active site in the naturally occurring peptide. This is done by combining biological activity and structural studies (by means of absorption infrared (IR) and Raman spectroscopy (RS)) with the investigation of the peptide behavior at a liquid/solid interface, that is, a water/membrane35 interface (by means of nuclear magnetic resonance (NMR)) or a water/metal interface (by means of surface-enhanced Raman scattering (SERS)).36,37 There are numerous indications that proteinmetal interactions are significant in the fundamental understanding of interfacial phenomena38 and for a wide variety of technological applications, including microarrays,39 (bio)sensors,40 catalysis,41 and biocompatible coatings.42 In the area of protein screening, it is the first crucial step for obtaining a functional and structural understanding of the activity of many hormones, host-defense peptides and lipopeptides, and toxins, for clarification of the principles that govern molecular recognition between membrane-spanning polypeptides and binding/association at the membrane interface and for the formation mechanism of hard tissues, such as bone and teeth, or biomineralization.43 In this respect, various chemistry- and biology-based methods have been developed utilizing the biotinstreptavidin interaction,44 antigenantibody interactions,45 and proteinligand (or proteinprotein) interactions.46 By using these criteria, we have determined the vibrational structure and the adsorption mechanism of bombesin and its analogues on silver, gold, and copper surfaces (electrode and/or in sol). We have also determined the changes in this adsorption process with both the applied electrode potential and the type of mutations in the bombesin neuropeptide, its C-terminal different length (BNX-14) and 614 amino acid length (BN614) fragments, and related peptides.36,37,4754 The observed SERS 6710

dx.doi.org/10.1021/jp200805f |J. Phys. Chem. B 2011, 115, 6709–6721

The Journal of Physical Chemistry B signals were correlated with the contribution of the structural components of bombesin to its ability to interact with a bombesinpreferred metabotropic seven-transmembrane G-protein coupled receptor superfamily, rGRP-R. The present spectroscopic study encompasses human, pig, and frog neurotensins and four single-site mutants of human neurotensin having the following modifications: [Gln4]NT, [Trp11]NT, [D-Trp11]NT, and [D-Tyr11]NT. Table 1 presents the amino acid sequences of these neuropeptides. Because we are interested in the structural basis for the ligandreceptor interaction, our investigations are focused particularly on the adsorbed structures of the above-mentioned peptides deposited onto electrochemically roughened silver substrates, their likely adsorption mechanisms on this surface, and changes in this process due to natural amino acid replacement. This study also attempts to correlate these observed changes with changes in their biological activity due to the unique ability of these peptides to interact with NTR1. The SERS technique is used to address these questions because it is a simple and rapid method for probing different types of supramolecular architectures and for studying adsorption phenomena at the peptide level. These interactions are believed to be of great significance. Their significance has been attributed to the process of protein adsorption often being energetically favorable in the presence of a solid surface; the adsorption of proteins does not affect their binding capabilities, implying that their structures are not strongly perturbed on the surface. On an interface that is formed between a biomolecule and metal surface, proteins have regions that directly interact with this surface. The amino acid composition and sequence of these regions usually determine the adsorption behavior of proteins on given metal surfaces. Therefore, analysis of the SERS signal (enhancement, broadness, and wavenumber) coming from the constituents’ amino acids has been useful in understanding possible ways in which a peptide could interact with the surrounding medium, such as how a peptide binds at a solid/liquid interface.36,37,4754The aforementioned experiments were complemented by Fourier-transform Raman (FT-RS) and absorption infrared (FT-IR) spectroscopies for determining the vibrational structures of the nonadsorbed peptides. Thus, the FT-RS and FT-IR spectra of human, pig, and frog neurotensin and four mutants of human neurotensin, [Gln4]NT, [Trp11]NT, [D-Trp11]NT, and [D-Tyr11]NT, are also reported and briefly discussed in this work.

’ EXPERIMENTAL SECTION Neurotransmitters. Human, pig, and frog neurotensin and single-site mutants of human neurotensin, [Gln4]NT, [Trp11]NT, [D-Trp11]NT, and [D-Tyr11]BN, were purchased at Bachem Co., Switzerland. Their purity and chemical structures were proven by means of the 1H and 13C NMR spectra (Bruker Avance DRX 300 MHz spectrometer) and electrospray mass spectrometry (Finnigan Mat TSQ 700). FT-IR Measurements. Thin palettes containing ∼0.5 mg of each peptide dispersed in 200 mg of KBr were used for the infrared measurement. The spectra were recorded at room temperature as an average of 30 scans using a Bruker infrared spectrometer (model EQUINOX 55) equipped with a Nernst rod as the excitation source and a DTGS detector in the 4004000 cm1 range with the spectral resolution of 4 cm1. FT-Raman Measurements. FT-Raman measurements were performed for samples placed on a glass plate. FT-RS spectra

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were recorded on a Nicolet spectrometer (model NXR 9650) combined with a liquid-nitrogen-cooled germanium detector. Typically, 1000 scans were collected with the resolution of 4 cm1. Excitation at 1064 nm was used from a continuum-wave Nd3þ:YAG laser. SERS Measurements. To obtain a sufficiently enhanced intensity of the SERS bands, the silver substrates were electrochemically roughened before peptide adsorption. Roughening was carried out in a conventional three-electrode cell with a large platinum sheet as the counter electrode and a Ag/AgCl (1 M KCl) electrode as a reference (all potentials are given versus this electrode). The silver was roughened by three successive negativepositivenegative cycles in a 0.1 M KCl aqueous solution from 0.3 to 0.3 V at a sweep rate of 5 mV s1. The cycling was finished at 0.3 V, and then the silver electrode was maintained at 0.4 V for 5 min. Afterward, the working electrode was removed at an open circuit potential and carefully rinsed with water. Figure 1S (see the Supporting Information) gives an overview of the morphology of the roughened silver substrates used in this work. Raman spectra were recorded with an ISA T64000 (Jobin Yvon) Raman spectrometer equipped with Kaiser SuperNotchPlus holographic filters, a 600 groove/mm holographic grating, an Olympus BX40 microscope with a 50 long-distance objective, and a 1024 256 pixel nitrogen-cooled CCD detector. A Laser-Tech model LJ-800 mixed argon/krypton laser provided the excitation radiation at 514.5 nm. The laser power at the sample was set to 1 mW (∼104 W/cm2). The accuracy of frequency readings was (1 cm1. Before each series of the experiments, the system was calibrated using Si as a standard. Usually, the SERS spectra were measured from 10 spots of the electrochemically roughened silver surface that had before been immersed in a 0.1 mM solution of the peptide. The spectra from the series were almost identical (very well reproducible) except for small differences (up to ∼5%) in some band intensities. Thus, at the concentration of 104 M, we expect formation of monolayer, only. To prove this statement, we measured the SERS spectra for species adsorbed from 10 and 100 times more diluted solutions. This is a standard procedure in our laboratory. In these cases, the SERS spectra were identical with those presented in the paper; however, the S/N ratios were lower. From this reason, we present the SERS spectra of adsorbed species obtained from the concentration of 104 M. These spectra were analyzed further. No spectral changes that could be associated with sample decomposition or desorption processes were observed during these measurements. Generalized Two-Dimensional Correlation Analysis. G2D correlation analysis of the SERS spectra of single-site mutants of NT adsorbed on the electrochemically roughened silver substrate was performed using the software SpectraCorr, version 1.1 SP1, by Thermo Fisher Scientific Inc., 20042007. The [Trp11]NT and [D-Trp11]NT and human NT and [D-Tyr11]NT SERS spectra were normalized. Th G2D maps were then generated using variations of L-/D-amino acid conformations as a perturbation variable.

’ RESULTS AND DISCUSSION FT-RS and FT-IR Studies. Figures 1 and 2 show, for the first time, Raman (FT-RS) and absorbance (FT-IR) spectra of human, pig, and frog neurotensins and single-site mutants of human neurotensin, [Gln4]NT, [Trp11]NT, [D-Trp11]NT, and 6711



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Figure 1. FT-Raman spectra of (A) human, (B) pig, and (C) frog neurotensin and single-site mutants of human neurotensin, (D) [Gln4]NT, (E) [Trp11]NT, (F) [D-Trp11]NT, and (G) [D-Tyr11]NT, in the spectral range of 1750550 cm1.

[D-Tyr11]NT, in the wavenumber range of 1750550 cm1. Unfortunately, due to the very low amount of the abovementioned neurotransmitters, it was not possible to measure the FT-RS spectra of these molecules in aqueous solution. Therefore, the solid-state Raman spectra are shown, because it has been established and accepted that, in an aqueous solution, many proteins do not change their secondary structures,55,56 except in extreme pH conditions. As can be seen, the presented FT-RS spectra show more band numbers as compared with the absorbance spectra. Additionally, a comparison of these spectra reveals similar vibrational patterns that mainly correspond to the characteristic tyrosine residue modes listed in Table 1S (Supporting Information). Because the bands of these modes have been well-defined, they are not discussed in detail here.5759

An exemption is a pair of Tyr-Raman markers (Fermi doublet) at ca. 850/830 cm1 (Figure 1). The relative intensity ratio of this doublet has been correlated with the state of phenolic hydrogen bonding in globular proteins and para-phenoxyl model compounds, with a typical range from 0.3 (when OH is a strong hydrogen-bond donor) to 2.5 (strong acceptor).60 In the case investigated in this work, the peptides, excluding [D-Tyr11]NT, at 850/830 cm1 had a relative intensity ratio below 1 (see Figure 1), suggesting that the hydroxyl phenol group is hydrogen bonded to a negative acceptor, such as the COO group of Glu4 or Leu13, most probably from the neighboring molecule. The vibrational spectra of [Trp11]NT and [D-Trp11]NT additionally exhibited the W2 (∼1578 cm1), W3 (∼1550 cm1), W7 (∼1359/1340 cm1), W16 (∼1010 cm1), and W18 6712



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Figure 2. FT-IR spectra of (A) human, (B) pig, and (C) frog neurotensin and single-site mutants of human neurotensin, (D) [Gln4]NT, (E) [Trp11]NT, (F) [D-Trp11]NT, and (G) [D-Tyr11]NT, in the spectral range of 1750550 cm1.

(∼746 cm1) modes of the tryptophan residue, reflecting the amino acid composition in the investigated peptides. The native neurotensin sequence contains two L-tyrosine residues at positions 3 (Tyr3) and 11 (Tyr11). Tyr3 is replaced by L-histidine (His3) in frog NT, whereas in the case of the single-site mutated peptides [D-Tyr11]NT, [Trp11]NT, and [D-Trp11]NT, the L-tyrosine at position 11 was substituted by D-Tyr, Trp, and D-Trp. Several studies have examined the NT structure using NMR spectroscopy. However, these studies have led to three different conclusions. The first suggests that, when neurotensin is bound to NTR1, it exists in a compact structure with a proline type I turn in its Arg9Ile12 region.61 The second proposes that NT adopts a β-strand conformation.62 Meanwhile, the third concludes that NT behaves as a flexible random-coil in aqueous solution.63 However, with alcohol solvents (TFE and HFIP) and

DPC micelles that mimic the membrane environment, fulllength NT has an extended, though slightly bent, structure with a great adaptability of the N-terminus (residues from 1 to 7) to the environment and accessibility to the solvent.35 Figure 3 shows 14 possible conformations of NT in the DPC micelles.35 The disordered/bent peptide arrangement is also adopted by human, pig, and frog neurotensin and single-site mutants of human neurotensin, [Gln4]NT, [Trp11]NT, [D-Trp11]NT, and [D-Tyr11]NT, in the solid state, as can be deduced from spectra presented in Figures 1 and 2. The symmetric medium relative intensity Raman signals at 16701664 cm1 (Figure 1) and strong broad absorbance at 16691654 cm1 (Figure 2) fall in the range that is typical for the amide I band of the random coil/ bent secondary structure (see Table 1S in the Supporting Information for precise band positions).64 Consequently, the 6713



Figure 3. NMR solution structure of human neurotensin in a membrane-mimetic environment (RSCB Protein Data Bank, Id 2OYV.pdb, ref 35). Possible mechanism of NT interaction with the electrochemically roughened silver substrate.

15481534 cm1 infrared-active (amide II) and the 1253 1236 cm1 Raman-active (amide III) bands are indicative of the formation of these secondary structures (see Table 1S in the Supporting Information). Other bands that match well-characterized vibrations belong to the aliphatic Asn, Glu, Gln, Lys, and Arg side chain vibrations and show significant absorbance/enhancement. These vibrations are the strong asymmetric broad Raman band at ca. 1447 cm1 (Figure 1) and the medium-weak absorption band near 1455 cm1 (Figure 2). They are mainly due to deformations of the CH2 groups (δ(CH2)). The methylene group also gave rise to the ca. 1340, 1316, and 884 cm1 Raman spectral features with medium relative intensity that are assigned to its wagging, twisting, and rocking vibrations, respectively (see Table 1S in the Supporting Information for detailed band assignments). An alternative contribution to the vibrational spectra from the aforementioned nonaromatic amino acids came from the amino (16461623, ca. 1204, ca. 1182, 11501126, and 10721040 cm1) and carbonyl (ca. 1390, 722, 677668, and 596 cm1) groups of the polypeptide side chains (see Table 1S, Supporting Information). SERS Studies. Figure 4 compares the SERS spectra of human, pig, and frog neurotensins and single-site mutants of human neurotensin, [Gln4]NT, [Trp11]NT, [D-Trp11]NT, and [D-Tyr11]NT, adsorbed from ca. 104 M solution onto electrochemically roughened silver substrates. Table 2 lists the observed wavenumbers of the most important SERS signals of these spectra; conclusions can be drawn about the manner of these peptides’ adsorption onto the given substrate, proposing allocation to the normal mode motions. To assess the reliability of these assignments, detailed information was obtained through literature studies6567 and from the Raman spectra presented in this work (Figure 1). Variations in the enhancement, broadness, and wavenumber shift of the corresponding Raman and SERS bands coming from the constituents’ amino acids were analyzed. Taking into account previous studies that have shown that aromatic amino acids give rise to intense SERS spectra of many peptides and exclusively or predominantly dominate these spectra,36,37,4754,6567 it was expected that, in all of the SERS spectra presented in this work (Figure 4), spectral features due to aromatic amino acid vibrations would be mainly enhanced.

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Therefore, the modes of the two tyrosine residues (Tyr3 and Tyr11/D-Tyr11) of the sequence of human, pig, and human single-site [Gln4] and [D-Tyr11] neurotensins, the His3 and Tyr11 residues of the frog NT, and the Tyr3 and Trp11/ 11 D-Trp residues of [Trp11]NT and [D-Trp11]NT (Table 1) should determine the profile of the SERS spectrum of the proper analogue, under the requirement that a given residue interacts with the silver surface. As is evident from Figure 4, this was this case. The SERS spectra of human NT, pig NT, frog NT, [Gln4]NT, and [D-Tyr11]NT adsorbed on the electrochemically roughened silver substrates showed very similar SERS spectral features (Figure 4), that is, ca. 1500, ca. 1450, ca. 1430, ca. 1350, ca. 1270, ca. 1150, ca. 1072, ca. 1001, ca. 958, ca. 884, ca. 825, and ca. 610 cm1 (see Table 2 for detailed band wavenumbers). From these, the most noteworthy contribution at ca. 1500 cm1 (very strong, fwhm = 2023 cm1; fwhm denotes full width at half-maximum) was due to the Y19 (CC) ring mode (according to Willson numbering).68,69 The Y19 mode for the protonated Tyr residue is proposed to absorb with an uncommonly small signal at 15251518 cm1, whereas the deprotonated form at 14961486 cm1 was accompanied by the medium enhanced Y19b mode, which, in the SERS spectra presented in Figure 4, was detected at ca. 1430 cm1 (fwhm = 1419 cm1) (Table 2).69 Thus, we believe that these two bands are associated with the deprotonated form of Tyr. However, given the respective pKa value for the phenol hydroxy group of Tyr (10.10) and the pH conditions of our experiments (pH ≈ 8.5), Tyr should contain the protonated hydroxyl group. However, it would not be surprising for the positively charged silver surface at the open circuit to act as a Lewis acid and facilitate tyrosinate deprotonation. Strikingly, there was a similar relative intensity ratio of these two bands and a spectral feature at ca. 1450 cm1 in the human NT, pig NT, frog NT, [Gln4]NT, and [D-Tyr11]NT SERS spectra. Moreover, the enhancement only slightly decreases when going from human NT, frog NT, and [D-Tyr11]NT to pig NT and [Gln4]NT. The ca. 1450 cm1 band (fwhm = 1721 cm1) was due to the vibrations of the tyrosine β-methylene group.70 Either the overlap by the CH2 deformations of the other side chains or the change in the CH2/ silver substrate proximity probably led to the detected shift in this band wavenumber (1446 T 1453 cm1) between the SERS spectra of the five discussed peptides. This could be supported by a ca. 1350 cm1 band (fwhm = 1517 cm1) due to CH2 wagging motions that was slightly more intense in the SERS spectra than in the corresponding Raman spectra. Another band that exhibited pronounced enhancement in the spectra of the discussed peptides was a SERS signal at ca. 1270 cm1, attributed to the ν(phenol-O) (Y7a) and δ(CH) vibrations of deprotonated Tyr (Table 2).69,71 It had a comparatively narrow width (fwhm = 1416 cm1) that excluded the possibility of contributions from amide III motions, as suggested in the case of Tyr homodipeptides adsorbed on a colloidal silver surface.65 Hence, we believe that the very low 1240 cm1 SERS spectral feature for adsorbed pig NT and [Gln4]NT is due to the amide III mode. In the case of human, frog, and [D-Tyr11] NT, the spectral pattern in the region of 13001200 cm1 was slightly more complex than that for pig NT and [Gln4]NT, showing, instead of the 1240 cm1 SERS signal, two additional bands at ca. 1290 (shoulder at ca. 1270 cm1) and 1220 cm1 (Figure 4). This could reflect either a different environment or protonation state of the second Tyr residue in the case of human NT and [D-Tyr11]NT. However, in the sequence of frog NT, 6714



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Figure 4. SERS spectra of (A) human, (B) pig, and (C) frog neurotensin and single-site mutants of human neurotensin, (D) [Gln4]NT, (E) [Trp11]NT, (F) [D-Trp11]NT, and (G) [D-Tyr11]NT, in the spectral range of 1750550 cm1 adsorbed at the open-circuit potential on electrochemically roughened silver substrates. Measurement conditions: ∼104 M; excitation wavelength, 514.5 nm; power at sample, ∼1 mW. Insets: (A) expanded Raman intensity scale by a factor of 2 and (B) expanded Raman intensity scale by a factor of 4, excluding [Trp11]NT and [D-Trp11]NT.

there was only one tyrosine residue at position 11 (Tyr3 replaced by His3). Therefore, these two spectral features are due to ν(CC) þ δ(CβH2) and Ft(CH2) of Tyr.6972 When the ca. 1270 cm1 SERS signal was the strongest of the spectrum, the ca. 1240 cm1 band was slightly enhanced, as in the case of pig NT and [Gln4]NT.

If this band dropped in relative intensity (to around 9080% of that of the ca. 1500 cm1 band), the ca. 1290 and 1220 cm1 bands appeared, as in the case of adsorbed human NT, frog NT, and [D-Tyr11]NT. This could be explained by considering that the human NT, pig NT, frog NT, [Gln4]NT, and [D-Tyr11]NT 6715



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Table 2. Wavenumbers and Band Assignments for SERS Spectra of Human, Pig, and Frog Neurotensin and Single-Site Mutants of Human Neurotensin, [Gln4]NT, [Trp11]NT, [D-Trp11]NT, and [D-Tyr11]NT wavenumbers (cm1)

a

assignment

human NT

pig NT

frog NT

[Gln4]NT

[Trp11]NT

[D-Trp11]NT

[D-Tyr11]NT

SERS

SERS

SERS

SERS

SERS

SERS

SERS

bent and/or Fs(NH2) in N, R, and K

1632

Y8a or W8a Y8b or W8b

1617 1589

1618 1589

νaa(COO) in E and/or C-terminal δ(CβH2)Y, ν(CC)Y,

1541 1501

1633

1634

1623

1592

1597

1618 1585

1592 1568

1559

1542

1558

1542

1506

1503

1507

1617 1587 1534

and/or AII Y19 ν(CC) of Trp phenyl co-ring

1500 1503

1499

δ(CH2)

1449

1452

1450

1453

1449

Y19b, Fs(CβH2)Y,W, and/or δ(CH2)

1430

1433

1430

1434

1433

1445

1428

νa(COO) in E and/or C-terminal L W7 (Fermi resonance) and/or Fw(CH2)

1393

1392

1394

1395 1350

1393 1343

1388

Fw(CH2)

1348

1349

ν(CC)Y, Y7a (ν(CO)), ν(COH)Y, and δ(CβH2)Y,W

1291

Y7a (ν(CO)), δ(CH)Y, and/or ν(CC)Wphenol

1269

1272

1270

1273

1272

1272

Y7a (ν(CO)), Fb(COH)Y, Ft(CH2)Y,W, AIII, and/or W

1220

1239

1222

1241

1224

1239

1219

1181

1187

1351

1351

1289

1351 1295

Y9a (Fb(CH)), ν(CC), and/or Fr(CβγδH2) νas(CCN) and/or Ft(NH2) in N, R, and/or K

1145

1148

1147

1446

1150

Fb(CH)phenylW ν(CO)Y, ν(CC)trans alkyl chain, and/or Fb(CH)W

1288

1147 1136

Ft(NH2) of guanido group of R

1072

1074

1074

1076

Y and/or W16 (benzene and pyrrole ring

1001

1005

1002

1006

1002

958

959

960

963

962

1268

1143 1136 1085

1108

1068

1072

1003

1001

breathing out-of-phase) Y/W (ring bend) ν(CCdO), ν(CC), and/or Y (ring bend) Fr(CH2)

968 946

884

W18 δ(COO) in E and/or C-terminal L

884

885

888

889 746 708

713

W and/or Fr(COOH)

677

Fw(COO)

610

614

612

613

613

611

611

a

Abbreviations (residues): Y, tyrosine; W, tryptophan; R, arginine; N, asparagine; E, glutamic acid; L, leucine; and K, lysine. Abbreviations (vibrations): ν, stretching; δ, deformation; Fw, wagging; Fb, bending; Ft, twisting; Fr, rocking; Fs, scissoring; s, symmetric; as, antisymetric.

bond to the electrochemically roughened silver substrate through the oxygen atom of the deprotonated hydroxyl group of phenol and that the phenol ringO bond of adsorbed human NT, frog NT, and [D-Tyr11]NT shows a slight deviation from a parallel orientation with respect to the silver surface normal. Other features of tyrosine oscillations in the human NT, pig NT, frog NT, [Gln4]NT, and [D-Tyr11]NT SERS spectra that invite further discussion are the bands at ca. 1001 (fwhm = 1719 cm1), ca. 958 (fwhm = 1612 cm1), and ca. 830 cm1 (fwhm = 1715 cm1) (Table 2).6872 The two former bands appeared with low relative intensity in the SERS spectra (Figure 4), decreasing when going from pig NT to human NT, [Gln4]NT, and [D-Tyr11]NT to frog NT, but not in the Raman spectra (Figure 1). Although the origin of these signals is puzzling, we note a possible spectroscopic assignment to the ring breathing and bending modes of the adsorbed Tyr. The ca. 830 cm1 band appeared as a strong doublet at 850/830 cm1 in the Raman spectra (fwhm = 1315 cm1/ 2225 cm1) and had weak scattering in the SERS spectra (fwhm = 1816 cm1). However, the expanded SERS spectra

in Figure 4 (inset A) show the ca. 830 cm1 band to be a doublet with components near wavenumbers similar to those in the Raman spectra. Thus far, we have shown compelling evidence for interactions of the Tyr ring, the oxygen atom of the deprotonated hydroxyl phenol unit, and the CH2 group of human NT, pig NT, frog NT, [Gln4]NT, and [D-Tyr11]NT with the electrochemically roughened silver substrate. Additionally, these findings reflect analogous ways for these fragments to interact with the silver substrate. This consistency indicates that the following mutations, Tyr11 f D-Tyr11 in [D-Tyr11]NT, Glu4 f Gln4 in [Gln4]NT, Pro7 f Ser7 in pig NT, or Leu2-Tyr3-Glu4-Asn5 f Ser2-His3-Ile4-Ser5 and Pro7 f Ala7, are not significantly affected by the orientation of the human NT on the electrochemically roughened silver substrate, as has likewise been proposed for NTR1 (see the Introduction). No significant shifts in the wavenumbers or the broadness between the corresponding SERS and Raman bands of the tyrosine doublet were noted; this lack of change coincides with an indirect Tyr/silver surface interaction. 6716



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Figure 5. G2D-synchronous (left) and -asynchronous (right) correlation maps of the SERS spectra of human tryptophan single-site mutant NT adsorbed on a roughened Ag electrode as a function of the L and D conformation of the Trp11 residue; spectral ranges of (A) 17001200 and (B) 1200900 cm1.

To examine the effects of tyrosine at position 11 mutations on the neurotensin orientation (see the Introduction), we also determined

the adsorbed structures of [Trp11]NT and [D-Trp11]NT deposited onto the electrochemically roughened silver substrate. In the 6717



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Figure 6. G2D-synchronous (left) and -asynchronous (right) correlation maps of the SERS spectra of human NT adsorbed on a roughened Ag electrode as a function of the L and D conformation of the Tyr11 residue; spectral ranges of (A) 16001200 and (B) 1200900 cm1.

SERS spectra of these two peptides, the ca. 1500 cm1 (ν(CC) of the Trp phenyl co-ring), 1449 cm1 (δ(CH2)), 1433 cm1 (Fs(CβH2) of Trp), ca. 1350 cm1 (Fermi resonance of Trp

and/or Fw(CH2)), 1295 cm1 (δ(CβH2) of Trp), 1272 cm1 (ν(CC) of Trp phenyl co-ring), 1239 cm1 (amide III and/or ν(CC) of Trp phenyl co-ring), 1224 cm1 (Ft(CH2) of Trp), 6718


The Journal of Physical Chemistry B ca. 1002 cm1 (Trp phenyl co-ring stretch), and ca. 962 cm1 (Trp phenyl co-ring deformation) spectral features resemble those of human NT, pig NT, frog NT, [Gln4]NT, and [D-Tyr11]NT.49,52,73 These results agree well with the proposed mechanism of these peptides’ interaction with the electrochemically roughened silver substrate, mainly through the phenyl ring. However, some specific differences in the enhancement and position of these SERS signals were clearly observed. For example, for adsorbed [Trp11]NT, a few bands were slightly altered, having a change in intensity and wavenumber in comparison to that of human NT, pig NT, frog NT, [Gln4]NT, and [D-Tyr11]NT, which reflected a tilted orientation of the phenol co-ring of the Trp residue on the silver substrate, including those at 1503 (IV), 1239 (Iv), 1592 (Iv), and 1568 (Iv) cm1.This could be supported by the decrease in the W16 mode SERS enhancement with respect to the corresponding Raman spectrum. These variations were even more drastic for [D-Trp11]NT. Additionally, for this peptide, the 1272 and 1147 cm1 spectral features decreased considerably in relative intensity when compared with those exhibited by the other adsorbed peptides investigated in this work. However, the principal differences were the signals at 1239, 1181, 1136, and 1085 cm1, whose enhancements were not reproducible within the SERS spectra of human NT, pig NT, frog NT, [Gln4]NT, and [D-Tyr11]NT and were, therefore, assigned to the Trp residue lying flat on the electrochemically roughened silver substrate. These were due to the CC stretching and CH bending deformations of the phenyl co-ring of the Trp indole ring. A weak band at ca. 1072 cml in the SERS spectra for all of the investigated peptides was due to NH2 group twisting vibrations. The asymmetric CCN stretching vibration was enhanced as a medium intensity SERS signal at 11501143 cm1 (fwhm = 1417 cm1) in these spectra (see Table 2 for detailed band wavenumbers). Neither of these bands were seen in the corresponding Raman spectra (Figure 1), and their SERS relative intensities changed only slightly in the order of human NT > pig NT ≈ [D-Tyr11]NT > [Gln4]NT > frog NT. For the assumed orientation of the neurotensin, the νas(CCN) involves a large polarizability change in the direction of the surface normal, which explains the prominent enhancement of this mode. This would be expected if the peptide was bound to the surface through the amine group, with the CN bond axis tilted or perpendicular relative to the mean silver surface plane. In the case of [Trp11]NT and [D-Trp11]NT, it was difficult to estimate the scattering of νas(CCN). This was due to its overlap with the Fb(CH)phenyl W mode of the almost coincident wavenumber and could be overcome by applying generalized twodimensional correlation analysis (G2DCA). This method is useful in the analysis of spectral signals, which change as a function of many kinds of reasonable physical variables affecting the spectra, such as time, temperature, concentration, potential, pressure, conformation, and even chemical reaction.7476 Therefore, using the G2D correlation method and changes in L-/Damino acid conformation as a variable, we successfully distinguish these two modes, autopeaks at (1147, 1147) and (1136, 1136 cm1, in a G2D-synchronous correlation map (Figure 5B, left trace) generated from the SERS spectra of [Trp11]NT and [D-Trp11]NT adsorbed on the electrochemically roughened silver substrate and also detected the small dissimilarities in the profile of these SERS spectra. Both these autopeaks, and that at (1085, 1085) cm1, strongly imply that the enhancement of these bands changes most prominently with changes in the Trp conformer. Additionally, the

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(1136, 1136) cm1 autopeak varied more than those at (1085, 1085) and (1147, 1147) cm1. In addition to the autopeaks, two intensities at (1085, 1136) and (1085, 1181) cm1 and four medium intensities at (1136, 1181), (1002, 1147), (968, 1136), and (968, 1085) cm1 had positive cross-peaks present in the G2D-synchronous correlation map. The positive sign of these cross-peaks indicates that all of these SERS signals underwent conformation-dependent enhancement changes in the same direction. They increased in relative intensity for [D-Trp11]NT. Additionally, the G2D-asynchronous correlation map developed three prominent cross-peaks (Figure 5B, right trace). The appearance of these peaks in the G2D-asynchronous correlation map suggests that the directions of the transition moments of these modes are different. The positive sign of the (1085, 1147) and (1136, 1147) cm1 peaks indicates that spectral changes took place earlier at 1085 and 1136 cm1 than at 1147 cm1. The negative sign of the peak at (1147, 1181) cm1 suggests that spectral changes took place earlier at 1181 cm1 than at 1147 cm1. This leads us to the conclusion that alternation of the orientation of the Trp phenyl co-ring on the silver substrate slightly determines the strength of coordination of the guanidine group to this substrate. However, it does not disturb the COO/silver interactions, as is evident from the lack of auto- and cross-peaks due to the carboxyl group vibrations in the G2D correlation maps (Figure 6A). On the other hand, the reorientation of the deprotonated Tyr11 of human NT with respect to the roughened silver substrate upon L-isomer f D-isomer exchange ([D-Tyr11] single-site mutant of NT) slightly alters the strength of both the COO/silver substrate and the guanidine group/silver substrate interactions. This is evident from the G2D-synchronous correlation map presented in Figure 6 (left traces), which consists of one strong and two weak autopeaks at (1539, 1539) cm1 and (1388, 1388) and (1145, 1145) cm1, respectively, assignable to the carboxyl and guanidine groups' motions. The strong intensity of the first autopeak is not surprising because it overlaps with the out-of-plane mode of the Tyr ring that gains intensity upon increase of the tilt angle of the Tyr ring with respect to the silver surface normal. This means that the Tyr ring of human NT is slightly less tilted with respect to the surface normal that that of [D-Tyr11]NT. Human NT, pig NT, [Trp11]NT, [D-Trp11]NT, and [D-Tyr11]NT consist of two carboxyl groups, of glutamic acid at position 4 of the amino acid sequence and C-terminal leucine, under the pH conditions of the SERS experiment, while Glu4 is replaced by Ile4 and Gln4 in frog NT and [Gln4]NT, respectively. Therefore, the Leu13OH carboxyl moiety of frog NT and [D-Trp11]NT attaching to the positively charged silver surface could give rise to COO stretching. In the [Gln4]NT SERS spectrum, we observed a medium enhanced 1394 cm1 (symmetric) and a negligibly scattered 1557 cm1 (antisymmetric) band of this moiety that were less obvious in the SERS spectrum of frog NT (Figure 4, inset B). The split between the signal positions of νs(COO) and νas(COO) was sensitive to the mode of surface coordination of the carboxylate group. A value of 164 cm1 indicates that the carboxylate groups of Leu13OH form a bidentate structure.77 By analogy, we suggest that the medium relative intensity 13951388 cm1 SERS signal for the remaining peptides, decreasing from [Trp11]NT, [D-Tyr11]NT, [Gln4]NT, pig NT, and human NT to frog NT, comes from Leu13COO and reflects slightly different strengths of interactions with the silver substrate. The ca. 713 and ca. 611 cm1 spectral features could also be due to deformations and wagging vibrations of this group. 6719


The Journal of Physical Chemistry B However, for pig NT and [Gln4]NT, the slight scattering at ca. 1240 cm1, previously assigned to the amide III mode of the polypeptide backbone, may not have contributed to the SERS spectra presented in Figure 4. In addition, a variation at 16331617 cm1 occurred in the characteristic spectral range for the δ(NH2) mode of the arginine side chains rather than for amide I (Figure 4, inset B). The lack of amide modes in the SERS spectra may indicate either that the amide group was parallel to the mean surface plane or that it was too far from the surface to be enhanced.

’ CONCLUSIONS The results described above demonstrate the feasibility of using SERS spectroscopy to probe peptidemetal interactions that, to some extent, may mimic the mechanism of a substrate binding to its receptor. The SERS spectra of human, pig, and frog neurotensins and single-site mutants of human neurotensin, [Gln4]NT, [Trp11]NT, [D-Trp11]NT, and [D-Tyr11]BN, adsorbed on electrochemically roughened silver substrates differed considerably and exhibited features that could be used to identify the specific residues in the adsorbed peptide. By analyzing the position and broadness of the enhanced bands caused by the vibrations of these specific molecular fragments, an adsorption mechanism on this surface was proposed for human, pig, and frog neurotensin and single-site mutants of human neurotensin, [Gln4]NT, [Trp11]NT, [D-Trp11]NT, and [D-Tyr11]BN. The SERS spectra of all of the compounds investigated in this work demonstrate that the peptides deposited onto an electrochemically roughened silver surface show bands due to vibrations of moieties that were in close proximity to the silver substrate and thus should be located on the same side of the polypeptide backbone. These include the Tyr ring and the oxygen atom of the deprotonated hydroxyl phenol group or the Trp11 phenyl co-ring π-system, the CH2 group, the guanidine group of arginine, and the carboxyl group of C-terminal Leu. As was evident from the solution structure of neurotensin in the membrane-mimetic environments (Figure 3), only three positions in the sequence fit these requirements: 9, 11, and 13 (Arg9, Tyr11/Trp11, and Leu13). These residues interact with the silver substrate to constrain the rather rigid structure of the 913 C-terminal fragment, whereas the low affinity of the “binding sites” of the N-terminal fragment could be the consequence of a higher degree of exposure of the N-termini to the solvent. Therefore, significant changes were not observed in the adsorption mechanism for the following mutations: Glu4 f Gln4 in [Gln4]NT, Pro7 f Ser7 in pig NT, Leu2-Tyr3-Glu4-Asn5 f Ser2-His3-Ile4-Ser5, and Pro7 f Ala7. This observation implies that the N-termini of NT does not influence the adsorption mechanism on a roughened silver substrate similar as is not essential for interaction with NTR1 (see the Introduction). The observed SERS signals correlated well with the contribution of the structural components to the ability of these peptides to interact with NTR1. For example, the SERS spectra revealed that the substitution of native amino acids in these molecules with other ones changes their adsorption state slightly on an electrochemically roughened silver surface. Human, pig, and frog neurotensin and [Gln4]NT and [D-Tyr11]NT tend to adsorb on this surface via the tyrosine ring and the oxygen atom of the deprotonated phenol group of Tyr11, whereas the CH2 unit(s), most probably of Tyr11, Arg9, and/or Leu13, are in close proximity (not bound to) to the silver substrate. The observed

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changes in the enhancement of the deprotonated Tyr residue SERS signals further indicate the parallel orientation of a phenolO bond in regard to the silver surface normal for pig NT, [Gln4]NT, and [D-Tyr11]NT, whereas the orientation was slightly tilted for human and frog NT. In the case of [Trp11]NT and [D-Trp11]NT, the formation of a peptide/Ag complex was confirmed by strong SERS bands involving the phenyl co-ring of Trp11/D-Trp11 and CH2 vibrations, pointing to a tilted and flat orientation with respect to the surface substrate, respectively. This could also confirm the key role of the phenyl ring in recognizing the electrochemically roughened silver substrate and receptor pathway (see the Introduction). The above-mentioned spectral features were accompanied by a SERS signal caused by vibrations of the carboxyl group of C-terminal Leu13 and the guanidine group of Arg9. It can be concluded, based on these biological activity studies, that the free carboxyl group of Leu13 and the guanidine group of Arg9 are responsible for both the adsorption mechanism on the electrochemically roughened silver substrate and receptor recognition (see the Introduction).

’ ASSOCIATED CONTENT

bS

Supporting Information. Figure 1S1: scanning electron microscopy (SEM) images of the roughened silver substrate used in this work. Measurement conditions: (A) 15.0 kV 100k SE, scale 500 nm; (B)15.0 kV 20k SE, scale 20 μm. Table 1S: wavenumbers and band assignments for FT-Raman and FT-IR spectra of human, pig, and frog neurotensin and single-site mutants of human neurotensin, [Gln4]NT, [Trp11]NT, [D-Trp11]NT, and [D-Tyr11]NT. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: þ48-12-663-2077. Fax: þ48-12-634-0515.

’ ACKNOWLEDGMENT This work was supported by the State Department for Scientific Research of the Ministry of Science and Higher Education (Grant No. N N204 159136 to E.P.). Y.K. gratefully acknowledges HUFS for financial support. The authors are grateful to Prof. Maria Janik-Czachor and Dr. Marcin Pisarek from the Institute of Physical Chemistry, Polish Academy of Sciences, for the SEM image of the roughened silver surface. ’ REFERENCES (1) Shegal, I.; Powerst, S.; Huntley, B.; Powis, B.; Pitrelkowi, M.; Maihele, N. J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4673. (2) Carraway, R.; Leeman, S. E. J. Biol. Chem. 1973, 248, 6854. (3) Martin, S.; Botto, J.-M.; Vincent, J.-P.; Mazella, J. Mol. Pharmacol. 1999, 55, 210. (4) Carraway, R.; Leeman, S. E. J. Biol. Chem. 1976, 251, 7045. (5) Kalivas, P. W.; Richardson-Carlsonn, R.; Duffy, P. J. Pharmacol. Exp. Ther. 1986, 238, 1126. (6) Vincent, J. P.; Mazella, J.; Kitabgi, P. Trends Pharmacol. Sci. 1999, 20, 302. (7) Dubuc, I.; Nouel, D.; Coquerel, A.; Menard, J. F.; Kitabgi, P. Eur. J. Pharmacol. 1988, 151, 117. (8) Osbahr, A. J., III; Nemeroff, C. B.; Manberg, P. J.; Prange, A. J., Jr. Eur. J. Pharmacol. 1979, 54, 299. 6720


The Journal of Physical Chemistry B (9) Carraway, R. E.; Demers, L.; Leeman, S. E. Fed. Proc. 1973, 1. (10) Coquerel, A.; Dubuc, I.; Kitabgi, P.; Costentin, J. Neurochem. Int. 1988, 12, 361. (11) Feurle, G. E.; Muller, B.; Rix, E. Gut 1987, 28, 19. (12) Carraway, R. E.; Plona, A. M. Peptides 2006, 27, 2445. (13) Evers, B. M. Peptides 2006, 27, 2424. (14) Lazarus, L. H.; Brown, M. R.; Perrin, M. H. Neuropharmacology 1977, 16, 625. (15) Myers, R. A.; Shearman, M.; Kitching, M. O.; Ramos-Montoya, A.; Neal, D. E.; Ley, S. V. Chem. Biol. 2009, 4, 503. (16) Chavatte, K.; Terriere, D.; Jeannin, L.; Iterbeke, K.; Briejer, M.; Schuurkes, J.; Mertens, J. J. R.; Bruyneel, E.; Tourwe, D.; Leysen, J. E.; Bossuyt, A. J. Labelled Compd. Radiopharm. 1999, 42, 423. (17) Boeijen, A.; van Ameijde, J.; Liskamp, R. M. J. Org. Chem. 2001, 66, 8454. (18) Kozikowski, A. P.; Dodd, D. S.; Zaidi, J.; Pang, Y.-P.; Cusack, B.; Richelson, E. J. Chem. Soc., Perkin Trans. 1 1995, 1615. (19) Tanaka, K.; Masu, M.; Nakanishi, S. Neuron 1990, 4, 847. (20) Mazella, J.; Botto, J. M.; Guillemare, E.; Coppola, T.; Sarret, P.; Vincent, J. P. J. Neurosci. 1996, 16, 5613. (21) Chalon, P.; Vita, N.; Kaghad, M.; Guillemot, M.; Bonnin, J.; Delpech, B.; Le Fur, G.; Ferrara, P.; Caput, D. FEBS Lett. 1996, 386, 91. (22) Mazella, J.; Zs€urger, N.; Navarro, V.; Chabry, J.; Kaghad, M.; Caput, D.; Ferrara, P.; Vita, N.; Gully, D.; Maffrand, J. P.; Vincent, J. P. J. Biol. Chem. 1988, 273, 26273. (23) Dubuc, I.; Sarret, P.; Labbe-Jullie, C.; Botto, J. M.; Honore, E.; Bourdel, E.; Martinez, J.; Costentin, J.; Vincent, J. P.; Kitabgi, P.; Mazella, J. J. Neurosci. 1999, 19, 503. (24) Pettibone, D. J.; Hess, J. F.; Hey, P. J.; Jacobson, M. A.; Leviten, M.; Lis, E. V.; Mallorga, P. J.; Pascarella, D. M.; Snyder, M. A.; Williams, J. B.; Zeng, Z. J. Pharmacol. Exp. Ther. 2000, 300, 305. (25) Gully, D.; Canton, M.; Boigegrain, R.; Jeanjean, F.; Molimard, J. C.; Poncelet, M.; Gueudet, C.; Heaulme, M.; Leyris, R.; Brouard, A.; Pelaprat, D.; Labbe-Julie, C.; Mazella, J.; Soubrie, P.; Maffrand, J.; Rostene, W.; Kitabgi, P.; Le Fur, G. L. Proc. Natl. Acad. Sci. U.S.A. 1993 90, 65. (26) Kitabgi, P.; Carraway, R.; Van Rietschoten, J.; Granier, C.; Morgat, J. L.; Menez, A.; Leeman, S.; Freychet, P. Proc. Natl. Acad. Sci. U.S.A. 1997, 74, 1846. (27) Granier, C.; van Rietschoten, J.; Kitabgi, P.; Poustis, C.; Freychet, P. Eur. J. Biochem. 1982, 124, 117. (28) Carraway, R. E.; Leeman, S. E. In Peptides: Chemistry, Structure and Biology; Walter, R., Meinhofer, J., Eds.; Ann Arbor Science: Ann Arbor, MI, 1975; pp 679685. (29) Lazarus, L. H.; Perrin, M. H.; Brown, M. R. J. Biol. Chem. 1997, 252, 7174. (30) Rivier, J. E.; Lazarus, L. H.; Perrin, M. H.; Brown, M. R. J. Med. Chem. 1977, 20, 1409. (31) Quirion, R.; Regoli, D.; Rioux, F.; St-Pierre, S. Br. J. Pharmacol. 1980, 69, 689. (32) Loosen, P. T.; Nemeroff, C. B.; Bissette, G.; Burnett, G. B.; Prange, A. J.; Lipton, M. A. Neuropharmacology 1978, 17, 109. (33) Jolicoeur, F. B.; Barbeau, A.; Rioux, F.; Quirion, R.; St. Pierre, S. Peptides 1981, 2, 171. (34) Folkers, K.; Chang, D.; Humphries, J.; Carraway, R.; Leeman, S. E.; Bowers, C. Y. Biochemistry 1976, 73, 3833. (35) Coutant, J.; Curmi, P. A.; Toma, F.; Monti, J.-P. Biochemistry 2007, 46, 5656. (36) Podstawka, E. Biopolymers 2008, 89, 506. (37) Podstawka, E. J. Raman. Spectrosc. 2008, 39, 1290. (38) Weaver, M. J.; Zou, S.; Chan, H. Y. H. Anal. Chem. 2000, 72, 38A. (39) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760. (40) Yonzon, Ch. R.; Haynes, Ch. L.; Zhang, X.; Walsh, J. T., Jr.; Van Duyne, R. P. Anal. Chem. 2004, 76, 78. (41) Tada, H.; Bronkema, J.; Bell, A. T. Catal. Lett. 2004, 92, 93. (42) Li, H.; Sun, J.; Cullum, B. M. Nanobiotechnology 2006, 2, 17. (43) Volny, M.; Elam, W. T.; Ratner, B. D.; Turecek, F. J. Biomed. Mater. Res., Part B 2006, 80B, 505.

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(44) Pradier, C. M.; Salmain, M.; Zheng, L.; Jaouen, G. Surf. Sci. 2002, 502/503, 193. (45) Grubisha, D. S.; Lipert, R. J.; Park, H.-Y.; Driskell, J.; Porter, M. D. Anal. Chem. 2003, 75, 5936. (46) Hodneland, C. D.; Lee, Y.-S.; Min, D.-H.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5048. (47) Podstawka, E.; Ozaki, Y. Biopolymers 2008, 89, 807. (48) Podstawka, E.; Ozaki, Y.; Proniewicz, L. M. Langmuir 2008, 24, 10807. (49) Podstawka, E.; Ozaki, Y. Biopolymers 2008, 89, 941. (50) Podstawka, E. Biopolymers 2008, 89, 980. (51) Podstawka, E.; Proniewicz, L. M. J. Phys. Chem. B 2009, 113, 4978. (52) Podstawka, E.; Niaura, G. J. Phys. Chem. B 2009, 113, 10974. (53) Podstawka, E.; Niaura, G.; Proniewicz, L. M. J. Phys. Chem. B 2010, 114, 1010. (54) Podstawka-Proniewicz, E.; Niaura, G.; Proniewicz, L. M. J. Phys. Chem. B 2010, 114, 5117. (55) Dong, A.; Meyer, J. D.; Kendrick, B. S.; Manning, M. C.; Carpenter, J. F. Arch. Biochem. Biophys. 1996, 334, 406. (56) Eker, F.; Cao, X.; Nafie, L.; Schweitzer-Stenner, R. J. Am. Chem. Soc. 2002, 124, 14330. (57) DeLange, F.; Klaassen, C. H. W.; Wallace-Williams, S. E.; Bovee-Geurts, P. H. M.; Liu, X. M.; DeGrip, W. J.; Rothschild, K. J. J. Biol. Chem. 1998, 273, 23735. (58) Kim, S.; Barry, B. A. Biophys. J. 1998, 74, 2588. (59) Matsuno, M.; Takeuchi, H.; Overman, S. A.; Thomas, G. J., Jr. Biophys. J. 1998, 74, 3217. (60) Siamwiza, M. N.; Lord, R. C.; Chen, M. C.; Takamatsu, T.; Harada, I.; Matsuura, H.; Shimanouchi, T. Biochemistry 1975, 14, 4870. (61) Pang, Y. P.; Cusack, B.; Groshan, K.; Richelson, E. J. Biol. Chem. 1996, 271, 15060. (62) Luca, S.; White, J. F.; Sohal, A. K.; Filippov, D. V.; van Boom, J. H.; Grisshammer, R.; Baldus, M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 10706. (63) Barroso, S.; Richard, F.; Nicolas-Etheve, D.; Reversat, J. L.; Bernassau, J. M.; Kitabgi, P.; Labbe-Jullie, C. J. Biol. Chem. 2000, 275, 328. (64) Haris, P. I.; Severcan, F. J. Mol. Catal. B: Enzym. 1999, 7, 207. (65) Herne, T. M.; Ahern, A. M.; Garrell, R. L. J. Am. Chem. Soc. 1991, 113, 846. (66) Lee, H. I.; Kim, M. S.; Suh, S. W. Bull. Korean Chem. Soc. 1988, 9, 218. (67) Stern, D. A.; Salaita, G. N.; Lu, F.; McCargar, J. W.; Batina, N.; Frank, D. G.; Laguren-Davidson, L.; Lin, Ch.-H.; Walton, N.; Gui, J. Y.; Hubbard, A. T. Langmuir 1988, 4, 111. (68) Wilson, E. B., Jr.; Decius, J. C.; Cross, P. C. The Theory of Infrared and Raman Vibrational Spectra; McGraw-Hill: New York, 1955. (69) Hellwig, P.; Pfitzner, U.; Behr, J.; Rost, B.; Pesavento, R. P.; v. Donk, W.; Gennis, R. B.; Michel, H.; Ludwig, B.; Mantele, W. Biochemistry 2002, 49, 9116. (70) Venyaminov, S. Y.; Kalnin, N. N. Biopolymers 1990, 30, 1259. (71) Pieridou, G. K.; Hayes, S. C. Phys. Chem. Chem. Phys. 2009, 11, 5302. (72) Kim, J. E.; Pan, D.; Mathies, R. A. Biochemistry 2003, 42, 5169. (73) Cao, X.; Fischer, G. J. Phys. Chem. A 1999, 103, 9995. (74) Chalmers, J., Griffiths, P., Eds. Handbook of Vibrational Spectroscopy; Wiley: New York, 2002. (75) Dluhy, R.; Shanmukh, S.; Morita, S. I. Surf. Interface Anal. 2006, 38, 1481. (76) Noda, I. Appl. Spectrosc. 1993, 47, 1329. (77) Nakamoto, K. Infrared and Raman Spectra of Inorganic and Coordination Compounds, 4th ed.; Wiley & Sons: New York, 1986.

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