4978
J. Phys. Chem. B 2009, 113, 4978–4985
The Orientation of BN-Related Peptides Adsorbed on SERS-Active Silver Nanoparticles: Comparison with a Silver Electrode Surface Edyta Podstawka*,† and Leonard M. Proniewicz‡ Regional Laboratory of Physicochemical Analysis and Structural Research, Faculty of Chemistry, Jagiellonian UniVersity, ul. Ingardena 3, 30-060 Krakow, Poland, and Chemical Physics DiVision, Faculty of Chemistry, Jagiellonian UniVersity, ul. Ingardena 3, 30-060 Krakow, Poland ReceiVed: December 15, 2008; ReVised Manuscript ReceiVed: January 19, 2009
We used surface-enhanced Raman scattering (SERS) to characterize the adsorption behavior of bombesin (BN) and five BN-related peptides, including phyllolitorin, [Leu8]phyllolitorin, neuromedin C (NMC), neuromedin B (NMB), and PG-L (Pseudophryne guntheri), in a silver colloidal solution. Our experiments show that the pyrrole coring of the Trp and aromatic ring of Phe of these peptides are preferentially adsorbed on silver nanoparticles. However, the geometry of the rings and the strength of the interactions with this surface vary among peptides. Additionally, these peptides are weakly coordinated to the colloidal silver surface through the CdO fragment of a peptide bond, between Gln/Leu/His and Trp residues, and CsNsC and SsC fragments. Also, using the recently reported SERS spectra of these peptides immobilized onto an electrochemically roughened silver electrode surface, we demonstrate substrate-induced changes in the adsorption behavior of these peptides. Comparative analysis indicates that the interactions between peptides and the enhancing surfaces depend strongly on the geometry of the Trp, sCONHs, and SsC fragments of these biomolecules etched on the surfaces. Introduction During the last three decades, several bombesin-related peptides have been characterized, including phyllolitorin and its Leu8 analogue from Phyllomedusa sauVagei ([Leu8]phyllolitorin, L-phenylalanine at the 8 position (Phe8) of the phyllolitorin amino acid sequence substituted by L-leucine (Leu8)), neuromedin C (NMC, GRP18-27 - fragment from the 18th to 27th amino acid in sequence of gastrin releasing peptide), neuromedin B (NMB), and PG-L (Pseudophryne guntheri).1-6 The amino acid sequences of these peptides are listed in Table 1. The structures of BN, NMC, and NMB have been investigated using two-dimensional nuclear magnetic resonance (NMR)7-13 and vibrational14-17 spectroscopies in solid, water, and membranemimicking environments. From a structural point of view, the spectroscopic parameters obtained from these studies demonstrated that these peptides do not possess a defined structure in solution. On the other hand, in aqueous 2,2,2-trifluoroethanol (TFE) containing solution and phospholipids bilayer, they adopt a relaxed R-helical conformation, formed by the linear arrangement of three connected β-turns, in the C-terminal region, while their N-terminal part has a rather random structure. Side chain atoms of the three residues, Trp, His, and Leu residues of BN and NMC, which correspond to Trp, His, and Phe in NMB (see Table 1), which are important for the binding to the receptors18-22 and might play a key role in interacting with hydrophobic acyl chains of the phospholipids in the membrane, are oriented toward the same direction, rendering some amphipathic characters. Circular dichroism, fluorescence, and molecular dynamics simulation study support these results.14,23 Several reports also * To whom correspondence should be addressed. E-mail: podstawk@ chemia.uj.edu.pl. Phone: (48) 12-663-2077. Fax: (48) 12-634-0515). † Regional Laboratory of Physicochemical Analysis and Structural Research. ‡ Chemical Physics Division.
suggested that for these peptides there might be a conformational change to a β-turn/R-helix type structure upon binding to the receptors.24-26 The structures of the prohormone encoding phyllolitorins are similar to that of the aforementioned BNrelated peptides.27 These peptides function as neurotransmitters in the central nervous system2,6 and as regulators of numerous gastrointestinal functions;19,28 additionally, they play an important role in normal lung development and in several pathologic conditions in the lung.29,30 Furthermore, bombesin-related peptides are potent growth agents that cause proliferation of normal cells31 and can accelerate growth of various tumor cell lines (small lung cancers, prostate, gastric, pancreatic, colon, and breast carcinomas).32-36 The use of BN-related antagonists as carrier biomolecules for targeting cytotoxic drugs to tumor cells has also recently been proposed.37 However, little is currently known about the mechanism of action of these bombesin-related peptides. Given the structural similarity among these peptides and their receptors,38,39 it can be hypothesized that small alterations in both the amino acid composition and tertiary structures of these peptides may play an important role in determining their affinity to the mammalian bombesin/GRP-preferring subtype receptor (rGRP-R), neuromedin B-preferring subtype receptor (rNMBR), orphan subtype-3 receptor (hBRS-3R), and the subtype-4 receptor (fBB4).19-22 Therefore, to further explore the mechanisms of action, we examine the ability of these peptides to bind to a colloidal silver surface. We compare these results with the ability of these peptides to bind to an electrochemically roughened silver electrode surface.17 Previously, Podstawka et al.40-44 showed the applications of SERS spectroscopy for the determination of adsorbed molecular structures and changes in these structures and in the adsorption mechanism resulting from substitutions of the natural amino acids with synthetic ones of BN; its six modified analogues, including [D-Phe12]BN, [Tyr4]BN,[Tyr4,D-Phe12]BN,[D-Phe12,Leu14]BN,[Leu13--Leu14]-
10.1021/jp8110716 CCC: $40.75 2009 American Chemical Society Published on Web 03/18/2009
Orientation of BN-Related Peptides
J. Phys. Chem. B, Vol. 113, No. 14, 2009 4979
TABLE 1: Amino Acid Sequences of BN and Bombesin-Related Peptides compound amino acid position in BN sequence
amino acid sequence 1
2
3
4
bombesin (BN) pGlu- Gln- Arg- Leuphyllolitorin [Leu8]phyllolitorin (Phyllomedusa sauVagei) neuromedin B (NMB) neuromedin C (NMC, GRP18-27) PG-L (Pseudophryne guntheri) pGlu- Gly- Gly-
a
5
6
Gly- Asnp-Glup-GluGly- AsnGly- AsnGly- Pro-
7
8a,b
9a
10
11a
12b
13b
14a
GlnLeuLeuLeuHisGln-
TrpTrpTrpTrpTrpTrp-
AlaAlaAlaAlaAlaAla-
ValValValThrValVal-
GlyGlyGlyGlyGlyGly-
HisSerSerHisHisHis-
LeuPheLeuPheLeuPhe-
Met-NH2 Met-NH2 Met-NH2 Met-NH2 Met-NH2 Met-NH2
a Common amino acid in the sequence of all investigated peptides. b Amino acids oriented toward the same direction when interacting with hydrophobic acyl chains of the phospholipids in the membrane.
BN, and [Lys3]BN; and X-14 fragments (where X describes an amino acid from the 6h to 13th position in the amino acid sequence of BN), which were deposited onto a silver electrode and colloidal silver surfaces. On the basis of the current investigations and previously published data, the adsorption mechanism of these peptides is proposed. This is possible because the biomolecule has regions that directly interact with the metal surface. The amino acid composition and sequence of these regions usually determine the adsorption behavior of the peptides with respect to a given metal surface. Therefore, the analysis of enhancement, broadness, and wavenumber of the SERS signals coming from the constituent amino acids allows us to understand the ways in which a peptide can interact with the surrounding medium, including how a substrate binds to its receptor.17,40-48 Experimental Procedure Neurotransmitters. Bombesin-related peptides phyllolitorin and its Leu8 analogue from Phyllomedusa sauVagei ([Leu8]phyllolitorin, L-phenylalanine at the 8 position (Phe8) of the phyllolitorin amino acid sequence substituted by L-leucine (Leu8)) and PG-L (Pseudophryne guntheri) were purchased from Phoenix Pharmaceuticals Inc. (U.S.A.). Neuromedin B (NMB) and neuromedin C (NMC, GRP18-27 - a fragment from the 18th to the 27th amino acid of gastrin releasing peptide) were purchased from Bachem Co., Switzerland. SERS Measurements. AgNO3 and NaBH4 were purchased from Wako Co. (Japan) and used without further purification. Three batches of colloidal silver solutions were prepared using the standard procedure.49 Briefly, 8.5 mg of AgNO3 dissolved in 50 mL of deionized water at 4 °C was added dropwise to 150 mL of 1 mM NaBH4 immersed in an ice-bath with vigorous stirring. After the addition of AgNO3 was complete, the resulting pale-yellow solution was stirred continuously at 4 °C for approximately 1 h. The excitation spectra of the three batches of Ag solution prepared in this manner showed an absorbance maximum at around 396 nm. Aqueous sample solutions were prepared by dissolving the samples in deionized water. The concentration of the samples before mixing with the colloid was adjusted to 10-4 M. The freshly prepared sample solution was added to the silver solution such that the final sample concentration in the silver colloid was ∼10-5 M. The final pH was 8.3. The SERS spectra of the peptides were collected twice using each batch of the three silver colloids with a HoloSpec f/1.8i spectrograph (Kaiser Optical Systems Inc.) equipped with a liquid-nitrogen-cooled CCD detector (Princeton Instruments). The 785 nm line of a NIR diode laser (Invictus) was used as the excitation source. The laser power at the sample was set to ∼15 mW. All of the SERS spectra were recorded within 1 h of the sample having been added to the Ag solution. The spectra
obtained were almost identical (highly reproducible) except for small differences (up to ∼5%) in some band intensities. No spectral changes that could be associated with sample decomposition or desorption processes were observed during these measurements. Results and Discussion Figure 1 presents SERS spectra of BN, phyllolitorin, [Leu8]phyllolitorin, NMB, NMC, and PG-L adsorbed on a silver colloidal surface in an aqueous solution. The pH of the solution was around 8.3. Therefore, the amine and amide groups of these biomolecules were expected to be sNH2 and sCONH2, respectively, under the measurement conditions. As with previous data on the SERS spectra of BN and its 8-14 and 6-14 fragments adsorbed on colloidal silver nanoparticles,41,43,44 the assignment of the predominant SERS bands to normal coordinates was as follows: ∼1031 cm-1, Phe in-plane CH bending mode (ν18a); ∼1011 cm-1, Trp out-of-phase ring breathing mode (W16); ∼1005 cm-1, Phe symmetric ring breathing mode (ν12); ∼930 cm-1, CsCdO stretching mode; ∼880 cm-1, Trp (N1-H) stretching and Fermi resonance between phenyl ring breathing and out-of-phase ring bend overtone (W17); and ∼760 cm-1, Trp symmetric in-phase indole ring breathing mode. Additionally, the bands at 1690-1653 (amide I), 1280-1261 (amide III), ∼1400 (ν(CdO)), and 685-622 cm-1 (ν(CsS)) (see Table 2 for detailed band positions) matched the assigned spectral features as determined in earlier investigations by Podstawka et al.40,42 The allocation of the remaining SERS bands in these spectra is listed in Table 2. These band assignments agree with those of BN,15,40,41 its X-14 fragments (X - amino acids from the 6 to 13 position of the amino acid sequence of BN),42,44 and bombesin-related peptides.14,16,17 As is evident from Figure 1, there are differences in the enhancement and position of the aforementioned bands among the SERS spectra of bombesin-related peptides. These differences can be attributed to variations in the adsorption mechanism of these peptides that depend on the geometry of the biomolecules etched on the silver surface. Therefore, comparison with the constituent functional groups or fragments enables us to characterize important changes in the bonding properties of these systems. Previously, it was shown that the principal binding region of BN adsorbed on the colloidal silver surface is located on the L-tryptophane residue at the 8 position of its amino acid sequence (Trp8), which is located in a hydrophobic cavity.40 These SERS investigations also indicated simultaneous interactions between the phenyl and pyrrole corings and the silver nanoparticles in an edge-on orientation and with the pyrrole coring closer to the surface. In this orientation, the N1-H bond of the pyrrole coring is strongly coordinated to the Ag surface. Moreover, the detailed study of the BN SERS spectrum profile showed no interaction or only a weak interaction of the carbonyl
4980 J. Phys. Chem. B, Vol. 113, No. 14, 2009
Podstawka and Proniewicz
Figure 1. SERS spectra of BN, phyllolitorin, [Leu8]phyllolitorin, NMB, NMC, and PG-L adsorbed on a silver colloidal surface in the spectral range 1800-500 cm-1. Measurement conditions, ∼10-5 M; excitation wavelength, 785 nm; power at sample, ∼15 mW.
group with the colloidal silver surface; moreover, there was no interaction or only a weak interaction given the long distance at which the peptide bond between Gln7-Trp8 was placed with respect to the colloidal surface. Finally, no interaction or little interaction was found with the C-N bond arranged horizontally on this surface. These SERS signals were well correlated with the contribution of the structural components to the peptide’s ability to interact with the rGRP-R receptor (a G-protein-coupled receptor superfamily). The relative potency for inhibition of binding of 125I-[Tyr4]BN to rat pancreas acini cells was correlated with the behavior of the amide bond on the silver electrode surface.42 The amino acid sequence of NMC is analogous to the sequence of the Gln7 f His7 (substitution of L-glutamine (Gln) at the 7 position by L-histidine (His)) 5-14 C-terminal fragment of BN (see Table 1). Hence, it is expected that both peptides, BN and NMC, should exhibit similar SERS patterns on the basis of the following facts: (i) the first five amino acids of the BN N-terminus do not influence the SERS spectra of BN and its modified analogues immobilized onto the electrochemically roughened silver electrode surface40,42 as well as are not essential for BN high affinity to rGRP-R,50,51 although these amino acids contribute to the selectivity of the peptide for one or the other bombesin receptors;52 (ii) His is not involved in the adsorption of BN on the colloidal silver nanoparticles;41,43,44 and (iii) the
CdO moiety of the peptide bond between Gln7 and Trp8 of BN (rather than the CdO group of Gln7) and its arrangement are responsible for producing the increase/decrease in rGRP-R and silver surface affinities.14,42 Our results support these statements. However, the spectra of these peptides show differences in the positions and enhancement of certain bands. For example, the stronger enhancement of the amide III band, found at 1277 cm-1 for NMC versus the 1275 cm-1 band for BN, indicates that there is an intrinsic difference in the orientation of the peptide bond, occurring between the amino acids at the 7 position and Trp8, on the silver colloidal surface of the two peptides due to the substitution of Gln7 with a larger weakly basic, positively charged His7. This kind of amino acid replacement probably produces both a decrease in the angle formed between the NsH unit of the amide bond and the colloidal Ag surface (see Figure 1) and an increase in rGRP-R affinity.52 Therefore, we suggest that a structure-function correlation exists between the orientation of the sCONHs bond on the colloidal silver surface, which was proposed earlier for the modified 6-14 fragments of the BN amino acid sequence immobilized onto the electrochemically roughened silver electrode surface.42 Furthermore, the 1277 cm-1 band was shifted in wavenumber by -15 and 18 cm-1 and up-shifted in wavenumber by 13 and 19 cm-1 in the case of NMC and BN upon immobilization onto the electrochemically roughened and
ν(CdO) AI [β-turn/anti-parallel β-sheet/unordered] ν(CdO) in pGlu, Asn, and/or Gln, and/or δas(NH2) W1 [phenyl + pyrrole ν(N1sC8)] Phe (ν8a) W2 [phenyl], HisN1sH, and/or Phe (ν8b) W3 [pyrrole ν(C2dC3)] AII and/or W4 [ν(CdC)] His [ring stretch + δ(N1sH)] W5 and Fs(CH2) W6 [pyrrole (νs(N1C2C3) + δ(N1-H)) + phenyl δ(CH)], δas(CH3), and/or δ(CH2) ν(CdO) in pGlu, Asn, and/or Gln W7 [indole ν(N1sC8); Fermi resonance] and/or Fw(CH2) δi.p.(CH), Ft(CH2), W8 [ν(C3sC9) + δ(N1sH)], and/or His AIII [β-turn/β-sheet/random-coil] and/or His [CsH i.p. bend/ring breathing] W10 [ν(C3sC10) + ν(CsH)] and/or His [CH i.p. bend/ring breathing] ν(C-C)Trp and/or Phe (ν9a) Phe (ν7a) δ(N1sH) and/or Ft(NH2) in Asn and/or Gln ν(CsC)t alkyl chain and/or W13 ν(CsN), Ft(CH2), and/or His ν(CsC)t alkyl chain and/or Ft(CH2) Phe (ν18a) ν(CsC) W16 [phenyl and pyrrole o.o.p. ring breathing] Phe (ν12) ν(CsN), Fb(NH2), and/or phenyl Fb(CH) ν(CsC) ν(CsCdO) ν(CsC) W [o.o.p. CH phenyl deformation] and/or Fr(CH2) W17 [δ(N1sH) and Fermi resonance between phenyl ring breathing and o.o.p. ring bend overtone] ν(CsC) ν(CsC), νs(CNC) secondary amide, and/or His [ring o.o.p. bend] ν(CsC) W18 [sym phenyl/pyrrole i.p. ring breathing] and/or ν(CdO) in pGlu, Asn, and/or Gln W19 and/or ν(CsS) PCsT ν(CsS) PCsG ν(CsS) PHsT ν(CsS) PHsG and/or His
assignment
1397 1355
1394 1360/1344
1007
1011
763s
689
814 760
691
880
801
859
898
927
1004
1011s
1070 1032
1121
1206
835
880
933 902 888
967
1078
1071
987
1149 1136
1125
1225
675
780 742
803
936
1005
1079 1027
1172
1236
1243
1255
1294
1375 1352/1331
1430
1595 1574 1537
1613
electrode surface
1251 1234
1394 1356
1417
1544
1606
1637
colloidal surface
phyllolitorin
1275
1315, 1290
1454 1435
1590 1564 1508
1625
electrode surface
1438, 1424
1579 1552
1680, 1653 1622
colloidal surface
BN
628
706s, 689
759
830 807
886
887
932
1057 1009
1157 1127
1217
1233
1280
1298
1400 1356
1541
1643
colloidal surface
612
724
867
1015
1085
1146
1232
1282
1403 1363/1323
1461
1595 1572 1515
electrode surface
709 687, 661
785 761
831 807
878
946 926
1005
1012s
1031
1205 1159 1121
1249
1275
1405 1359/1343
1457 1422
1552
1603
1679
670
712
745
841
915
1022
1179 1204
1231
1265
1306
1392 1324
1590 1559 1529 1493 1466 1430
electrode surface
NMB colloidal surface
wavenumber (cm-1) [Leu ]phyllolitorin
8
708 686, 661
784 760
831
878
950 929 908
1011
1127 1105 1070
1188
1226
1277
1358/1345
1427
1549
1678
623
693
778 755
809
998
1018
1110
1170
1234
1249
1304
1375
1438
1575 1544 1527 1483
1634
electrode surface
NMC colloidal surface
TABLE 2: Wavenumbers and Band Assignments for SERS Spectra of BN and BN-Related Peptides Adsorbed on the Colloidal Silver Surfacea
685, 656 624
714
780 761
832
879
953 928
1007
1007
1069 1032
1205 1160 1123
1261
1408 1361/1343
1453 1429
1548
1666 1624
colloidal surface
724 700 681, 662
750
844 805
900
921
1002
1018
1084 1040
1152
1191
1236
1296
1350/1340
1421
1570 1545 1514 1498
1617
1744 1689
electrode surface
PG-L
Orientation of BN-Related Peptides J. Phys. Chem. B, Vol. 113, No. 14, 2009 4981
518 549
Phe (ν6b) W W Ft(CO) + δ(CdO) in pGlu, Asn, and/or Gln
assignment
Abbreviations: s, shoulder; ν, stretching; δ, deformation; Fb, bending; Fw, wagging; Ft, twisting; sym, symmetric; as, antisymmetric; i.p., in-plane vibrations; o.o.p., out-of-plane vibrations; t, trans conformation.
514 575 548 553 535 573 549
611 622 579 547 577 622 621
electrode surface colloidal surface
BN
TABLE 2: Continued
a
610
electrode surface colloidal surface electrode surface
NMC
colloidal surface electrode surface
NMB
colloidal surface electrode surface colloidal surface electrode surface
[Leu8]phyllolitorin
wavenumber (cm-1)
phyllolitorin
colloidal surface
PG-L
4982 J. Phys. Chem. B, Vol. 113, No. 14, 2009
Podstawka and Proniewicz colloidal silver surfaces, respectively. This indicates no substantial substrate-induced changes in the strength of the sCONHs/Ag interactions for these two peptides. However, the strength of these interactions was slightly weaker for BN than for NMC. A greater shift (∆ν ) -35 cm-1) in the amide I band position was reported for NMC immobilized onto the silver electrode surface,17 while, for NMC adsorbed on the colloidal silver nanoparticles, a 6 cm-1 upshift in wavenumber was observed (1669 cm-1 in the solid state f 1675 cm-1 on the colloidal Ag surface). This suggests that the deposition on the colloidal surface does not produce a superior change in the secondary structure of NMC that exists in a β-turn conformation in the nonadsorbed and adsorbed on the colloidal silver surface states. In contrast, for NMC immobilized onto the Ag electrode surface, it was reported that either sCONHs directly interacts with Ag without a change in the secondary structure or alternatively a possible change in the secondary structure of this peptide occurs from the β-turn for the nonadsorbed species to a hydrogen-bonded R-helical structure for this biomolecule adsorbed on the electrode. This is similar to what was proposed for BN adsorbed onto the electrochemically roughened silver electrode and colloidal silver nanoparticles surfaces,40,41 as well as that observed for BN attached to phospholipid bilayer membranes.14 For phyllolitorin, [Leu8]phyllolitorin, NMB, and PG-L, the substrate-induced changes in the relative intensities of the amide I and III bands are far more visible than the differences in the band positions. For example, a medium relative intensity of the amide I band is observed for NMB on the silver electrode, and no enhancement of this spectral feature was reported for phyllolitorin, [Leu8]phyllolitorin, and PG-L on this surface, due to the changes in the electron distribution of the amide bond.17 For all of these peptides adsorbed on the colloidal silver nanoparticles, the amide I band is weakly enhanced. Additionally, a considerable down-shift (∆ν ) ∼30 cm-1) in the amide I band wavenumber between nonadsorbed and adsorbed on the silver nanoparticles states is seen for phyllolitorin and [Leu8]phyllolitorin. For NMB (∆ν ) 7 cm-1) and PG-L (∆ν ) -8 cm-1) adsorbed on this surface, the observed shift in this band position is comparable to that for BN (∆ν ) -10 cm-1). One explanation for this behavior is that the -CONH- bond (-Leu-CONH-Trp-) of phyllolitorin and [Leu8]phyllolitorin binds without a change in the peptide secondary structure with the colloidal silver surface, adopting rather a parallel orientation on this surface, since the amide bands were expected to be enhanced only if the -CONH- group was not parallel to the surface.53 Alternatively, these adsorbed biomolecules may adopt the hydrogen-bonded R-helical structure. On the other hand, the -CONH- bond of NMB and PG-L adsorbed on the silver nanoparticles may accept a position that is slightly misaligned with the side of this surface. Hence, there is no direct -CONH-/Ag interaction for these peptides that preserves their solid state secondary structure (turn). The rather weak enhancement and position (1280-1261 cm-1) of the spectral feature due to the amide III vibrations supports the above statements. The most obvious Raman feature is the W16 (see Table 2 for detailed assignment) at 1006 cm-1, which decreases equally in relative intensity upon BN adsorption on the colloidal and electrochemically roughened silver surfaces and is somewhat up-shifted in wavenumber (∆ν ) 4 cm-1) for biomolecules adsorbed on the silver nanoparticles (1007 cm-1) in comparison to those immobilized onto the silver electrode surface (1011 cm-1). The wavenumber shift of this mode for [Leu8]phyllolitorin (∆ν ) 6 cm-1) and NMC (∆ν ) 7 cm-1)
Orientation of BN-Related Peptides adsorbed on the colloidal silver surface coincided within 2-3 cm-1 to that of BN, indicating that the π-electron system of the indole ring of these peptides does not interact directly with either silver surface and that the strength of interactions with both surfaces is similar. In addition, the band of this mode is slightly more enhanced for NMC adsorbed on the silver nanoparticles than for BN, for which the enhancement is in turn much stronger that that for [Leu8]phyllolitorin. This is in contrast to results obtained for the silver electrode surface that suggest the relative intensity decreases in the following order: [Leu8]phyllolitorin > BN > NC > NMB > PG-L ≈ phyllolitorin.17 This observation suggests that the angle formed between the indole ring and the silver nanoparticles surface decreases in the order NMC > BN > [Leu8]phyllolitorin. Thus, it decreases in the opposite direction for peptides deposited onto the electrochemically roughened silver electrode surface. In the case of NMB and PG-L adsorbed on the colloidal silver surface, the ν12 mode of the Phe residue has appreciable intensity around this wavenumber in the SERS spectra. On the other hand, it decreases in intensity in the phyllolitorin SERS spectrum. Also, there is almost no shift (∆ν ) 1-5 cm-1) or broadening (νfwhm ) 2 cm-1; fwhm, full width at half-maximum) for phyllolitorin, NMB, and PG-L. Additionally, the shape of this band was symmetric for PG-L, whereas, for phyllolitorin and NMB, it exhibited an asymmetric shape. This can be attributed, respectively, to the superposition of two bands (W16 and ν12) at coincidental wavenumbers (1009 and 1003 cm-1, respectively; wavenumbers obtained based on deconvolution of the 1007 cm1 band) and to the presence of the W16 mode (higher-wavenumber shoulder at 1011 and 1012 cm-1, respectively) that is hidden under the ν12 spectral feature. The aforementioned conclusions are supported by the enhancement of the other characteristic bands of Phe (i.e., ∼1606 cm-1, A1; ∼1205 cm-1, A1; ∼1032 cm-1, A1; and ∼622 cm-1, B2) (see Table 2 for detailed band positions and their allocations to normal coordinates) and Trp in the SERS spectra of phyllolitorin, NMB, and PG-L adsorbed on the colloidal silver surface. We note that the surface selection rules would predict that the normal modes with polarizability derivative components perpendicular to the surface should dominate the SERS spectra.54,55 It is therefore possible to determine the orientation of the Phe ring, assuming the C2V symmetry and that the molecular axes are such that z contains the C2 axis of symmetry and yz is the molecular plane with respect to the enhancing substrate. Applying this treatment to the presented SERS spectra, the B2 modes are expected to be large only when the ring is “standing up” on a metal surface. In this orientation, the A1 and B1 modes are also expected to exhibit good enhancement, while the vibrational modes of the A2 symmetry should not be apparent. On the other hand, for the Phe ring lying flat on the surface, only the A2, A1, and B1 modes should scatter effectively.56-60 Therefore, it can be concluded that the Phe ring of NMB and PG-L is slightly tilted from a perpendicular orientation with respect to the colloidal silver surface. However, for PG-L, the Phe ring is a little more removed from this surface in comparison to NMB. This is contrary to the results for these peptides deposited onto the electrochemically roughened silver electrode. In the case of phyllolitorin adsorbed on the silver nanoparticles, the comparable enhancement of the 1004 and 621 cm-1 bands is consistent with the conclusions either that the Phe ring is almost flat on this surface and one of the C-S bond conformers is enhanced at 621 cm-1 or that it adopts an almost perpendicular geometry with respect to the enhancing surface, being displaced somewhat from this surface. This behavior of the phyllolitorin Phe ring
J. Phys. Chem. B, Vol. 113, No. 14, 2009 4983 on the colloidal silver surface is consistent with the conclusions drawn for this peptide immobilized onto the silver electrode surface. Special attention should also be devoted to the ∼950 cm-1 spectral feature, whose behavior supports the predicted geometry of the Phe ring on the colloidal silver surface. This band is weakly enhanced for NMB and PG-L. For phyllolitorin, the band increases in intensity and shifts to a lower frequency by 9 cm-1 in comparison to the intensity and position in the SERS spectrum of the biomolecule adsorbed on the silver electrode surface.17 This band involves out-of-plane CCH deformations and is expected to decrease in intensity as the Phe ring lies less flat and simultaneously decrease in wavenumber due to a weakening of the bending force constant with increasing π-electron donation to the surface.60 There are other differences between the SERS spectra of BN, phyllolitorin, [Leu8]phyllolitorin, NMB, NMC, and PG-L that depend upon substrate exchange. The broad (fwhm ) ∼20 cm-1), intense band at ∼760 cm-1 (see Table 2 for detailed band positions and assignment), which is present in all of the SERS spectra of peptides adsorbed on the colloidal silver surface, except that of phyllolitorin (Figure 1), is negligibly enhanced in the SERS spectra of these biomolecules immobilized onto the silver electrode surface.17 Moreover, it is less pronounced for [Leu8]phyllolitorin adsorbed on the colloidal silver nanoparticles than for BN, NMB, NMC, and PG-L. This mode is, however, present as a low-wavenumber shoulder in the phyllolitorin spectrum. The selective enhancement of this mode implies that the pyrrole coring of these peptides, preferentially interacting with the silver nanoparticles, alters the orientation and strength of interactions, hence changing the relative intensity of this band in the following order: BN ≈ NMC > NMB ≈ PG-L > phyllolitorin > [Leu8]phyllolitorin. The observed shift in wavenumber by 0-3 cm-1 for the corresponding mode of the normal Raman spectra can be interpreted as evidence that there is no direct interaction between the π-electron system of the pyrrole coring and silver nanoparticles. It is known that SERS spectra show the selective enhancement of bands due to bond orientation relative to the metal surface. For this reason, the positions and relative intensities of the ∼880, ∼890, ∼1423, and ∼1530 cm-1 spectral features are informative of the N1sH/Ag, phenyl/Ag, C2dC3/Ag, and N1sC2dC3/ N1sH/Ag interactions, respectively. For the first band, the following changes of a medium enhancement, BN ≈ NMC ≈ PG-L > NMB > [Leu8]phyllolitorin > phyllolitorin, accompanied by almost no shift in wavenumber (∆ν ) 0-4 cm-1) can be attributed to a decrease in the strength of the indirect interaction between the silver nanoparticles and N1sH bond, for which a plane of BN, NMC, and PG-L adopts a tilted orientation with respect to the colloidal silver surface. Such an arrangement favors the interaction of the nitrogen atom’s lone pair of electrons with the metal surface. The next two bands, at ∼1530 and 1423 cm-1, in the SERS spectra of BN, NMB, NMC, and PG-L adsorbed on the colloidal silver surface are weakly enhanced, while, for phyllolitorin and [Leu8]phyllolitorin, these spectral features are hardly detectable. These are indicative that the N1sC2dC3 fragment of these biomolecules assists in their adsorption process on the colloidal silver surface. On the other hand, the medium and weak enhancement of the phenyl coring mode at around 890 cm-1 for BN and [Leu8]phyllolitorin adsorbed on the colloidal silver surface only indicates that the phenyl coring of these two peptides is slightly involved in the interaction with the silver particles.
4984 J. Phys. Chem. B, Vol. 113, No. 14, 2009 Another noteworthy aspect of the NMB, NMC, and PG-L SERS spectra (Figure 1) is the presence of three bands due to the rotational conformers of a C-S bond of L-methionine (Met). As is evident from the spectra, one PC-G and two PH-T conformations (notation introduced by Miazawa61 and Shimanuchi62) seem most preferable for NMB and NMC, whereas the one PC-T and two PH-T rotamers are preferable for PGL. The only one ν(C-S) conformer (PC-G) is found in the SERS spectra of BN and phyllolitorin adsorbed on the silver nanoparticles. In the case of the last peptide, [Leu8]phyllolitorin, two bands are observed in the wavenumber range corresponding to the C-S stretching region at 689 and 628 cm-1. According to the above discussion, these two bands undoubtedly correspond to the PC-G and two PH-G rotamers. However, for this peptide, a higher-wavenumber shoulder (at 706 cm-1, PC-G) on the strongly enhanced band at 689 cm-1 can also be detected. As may be deduced from the comparison of the SERS spectra recorded for these biomolecules adsorbed on the electrochemically roughened silver electrode and colloidal silver surfaces, the weak enhancement of the spectral features of the aforementioned conformers, except for the PC-G rotamers of [Leu8]phyllolitorin, indicates that the sulfur atom’s lone pair of electrons assists in the adsorption mechanism of these peptides, except [Leu8]phyllolitorin, on both enhancing surfaces. By contrast, the sulfur atom of [Leu8]phyllolitorin strongly binds to the colloidal silver nanoparticles. Moreover, the replacement of the electrode surface by the colloidal surface alters the conformations of the -CH2CH2S- fragment, hence shifting the wavenumbers exhibited by these rotamers. The SERS spectra obtained in the silver colloidal solution are also different from the spectra of peptides immobilized onto the electrochemically roughened silver electrode as a result of the enhancement of bands due to the ν(CdO) and ν(CsCdO) modes (see Figure 1 and Table 2 for band positions). The band of the former mode is one of the strongest bands for [Leu8]phyllolitorin, BN, and NMC deposited onto the silver electrode surface, and it exhibits weak relative intensity for phyllolitorin and NMB on this surface. On the other hand, it is weakly enhanced for all investigated peptides, except [Leu8]phyllolitorin (somewhat enhanced), adsorbed on the colloidal silver nanoparticles. This indicates that the CdO moiety is situated close to the colloidal silver surface and that the oxygen atom of these peptides attaches to the two surfaces in a different manner. The large magnitude of the upshift in wavenumber of this band for phyllolitorin, NMB, and NMC adsorbed on the silver nanoparticles in comparison to the electrode surface also indicates the different strength of these interactions. Further evidence for the substrate-induced changes in the CdO/Ag interactions comes from a variation in the enhancement of the CsCdO mode (at 926-933 cm-1), which can arise from the selective enhancement of this mode caused by an alternation in orientation of the CsCdO unit on both surfaces. One well resolved band at ∼832 cm-1 (see Table 2 for detailed band positions) due to the ν(C-C) + νs(CNC) mode is weakly enhanced in the SERS spectra of all the investigated peptides adsorbed on the colloidal silver surface only (Figure 1). Therefore, it can be used as an index of the peptides’ interaction through the nitrogen atom’s lone pair of electrons with this surface. It exhibits an insignificantly stronger relative intensity for BN than for [Leu8]phyllolitorin, NMB, NMC, and PG-L, whereas it is hidden under a strong very broad (fwhm ) 70 cm-1) band at 801 cm-1 for phyllolitorin. Therefore, we conclude that the C-N-C unit assists in the adsorption of these
Podstawka and Proniewicz peptides on the silver nanoparticles, while it does not play a role in the case of the silver electrode surface. Also, either the strength of this interaction is slightly stronger for BN than for remaining peptides or the C-N-C moiety for BN is closer to this enhancing substrate than for the other peptides. The 987 cm-1 spectral feature is weakly enhanced in the BN SERS spectrum only, supporting the above statements and suggesting that the C-N-C unit of BN approaches the colloidal silver surface with its N-C bond more normal to this surface than in the case of the other peptides; this allows easier access of the N-lone pair of electrons to the metal surface. Conclusions This paper reports our experiments on the adsorption behaviors of BN, phyllolitorin, [Leu8]phyllolitorin, NMB, NMC, and PG-L adsorbed on a silver colloidal surface in aqueous solution. These behaviors are compared to the results obtained for these peptides immobilized onto an electrochemically roughened silver electrode surface.17 According to the “surface selection rules”, as well as by analyzing and comparing the obtained SERS spectra, we conclude the following: (i) the adsorption behaviors of these peptides are different according to the enhancing substrate; (ii) the π-electron system of the indole ring of these peptides does not interact directly with either silver enhancing surface, and the strength of these interactions with both surfaces is similar; (iii) the angle formed between the indole ring and silver nanoparticle surfaces decreases in the order NMC > BN > [Leu8]phyllolitorin; thus, it decreases in the opposite direction than that for these peptides deposited onto the electrochemically roughened silver electrode surface; (iv) the pyrrole coring of these peptides preferentially interacts only with the silver nanoparticles, although both the strength of this interaction and the ring orientation vary among peptides; (v) contrary to the electrochemically roughened silver electrode, the Phe ring of NMB and PG-L on the colloidal silver surface interacts with this surface in a tilted-from-vertical orientation with respect to this surface; on the other hand, for phyllolitorin, the Phe ring is horizontal and nearly flat on the colloidal and electrode surfaces, respectively; (vi) there are intrinsic differences in the orientation of the peptide bond (-X-CONH-Trp-) on the silver colloidal surface and in the strength of the -CONH-/Ag interaction for these peptides adsorbed on the colloidal and electrode silver surfaces; (vii) the substrate-induced changes are also observed in the enhancement of bands due to the ν(CdO) and ν(CsCdO) modes and in the conformations of the sCH2CH2Ss fragment; (viii) only at the silver colloidal surface does the CsNsC fragment of all investigated peptides assist in the adsorption process of these biomolecules; however, for BN, the CsNsC unit occurs closer to this enhancing substrate than for the other peptides, approaching the colloidal silver surface with the NsC bond more normal to this surface than in the case of the other peptides. Except for the SERS signals due to the Phe ring and C-S bond vibrations, the aforementioned behavior of the investigated peptides adsorbed on the colloidal silver surface correlate well with the contribution of the structural components to the ability of these peptides to interact with the rGRP-R. However, there are no such exceptions in the case of these proteins deposited onto the electrochemically roughened silver electrode surface. Hence, it seems that the silver electrode surface is a more
Orientation of BN-Related Peptides selective enhancing surface than the colloidal one. Thus, the results presented here demonstrate the feasibility of using SERS spectroscopy to probe the protein-metal interactions that mimic the mechanism of a substrate binding to its receptor. Acknowledgment. This work was supported by the Polish State Committee for Scientific Research (Grant No. 1 T09A 112 30 to E.P.). References and Notes (1) Bevis, C. L.; Zasloff, M. Annu. ReV. Biochem. 1990, 59, 395. (2) Aguayo, S. M.; Kane, M. A.; King, T. E.; Schwarz, M. I.; Grauer, L.; Miller, Y. E. J. Clin. InVest. 1989, 84, 1105. (3) King, K. A.; Torday, J. S.; Sunday, M. E. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4357. (4) Ryan, R. R.; Katsuno, T.; Mantey, S. A.; Pradhan, T. K.; Weber, Ch. W.; Coy, D. H.; Battey, J. F.; Jensen, R. T. J. Pharmacol. Exp. Ther. 1999, 290, 1202. (5) Grinder, J. R. Am. J. Phys. Gastrointest. LiVer Physiol. 2004, 287, G1109. (6) Ohaki-Hamazi, H.; Iwabuchi, M.; Maekawa, F. Int. J. DeV. Biol. 2005, 49, 293. (7) Di Bello, C.; Gozzln, L.; Tonellato, M.; Corradini, M. G.; D’Auria, G.; Paolillo, L.; Trivellone, E. FEBS Lett. 1988, 237, 85. (8) Lee, S.; Kim, Y. FEBS Lett. 1999, 460, 263. (9) Carver, J. A. Eur. J. Biochem. 1987, 168, 193. (10) Carver, J. A.; Collins, J. G. Eur. J. Biochem. 1990, 187, 645. (11) Malikayi, J. A.; Edwards, J. V.; McLean, L. R. Biochemistry 1992, 31, 7043. (12) DI`az, M. D.; Fioroni, M.; Burger, K.; Berger, S. Chem.sEur. J. 2002, 8, 1663. (13) Reily, M. D.; Thanabal, V.; Omecinsky, D. O. J. Am. Chem. Soc. 1992, 114, 6251. (14) Erne, D.; Schwyzer, R. Biochemistry 1987, 26, 6316. (15) Carmona, P.; Lasagabaster, A.; Molina, M. Biochim. Biophys. Acta 1995, 1246, 128. (16) Carmona, P.; Molina, M.; Lasagabaster, A. Spectrochim. Acta., Part A 1995, 51, 929. (17) Podstawka, E. Biopolymers 2008, 89, 980. (18) Horwell, D. C.; Howson, W.; Naylor, D.; Osborne, S.; Pinnock, R. D.; Ratcliffe, G. S.; Suman-Chauman, N. Int. J. Pept. Protein Res. 1996, 48, 522. (19) Jensen, R. T. In Physiology of the Gastrointestinal Tract; Johnson, L. R., Jacobsen, E. D., Christensen, J., Alpers, D. H., Walsh, J. H., Eds.; Raven Press: New York, 1994; p 1377. (20) Ruff, M.; Schiffmann, E.; Terranova, V.; Pert, C. B. Clin. Immunol. Immunopathol. 1985, 37, 387. (21) Lebacq-Verheyden, A.-M.; Trepel, J.; Sausville, E. A.; Battey, J. F. In Handbook of Experimental Pharmacology; Sporn, M., Roberts, A., Eds.; Berlin: Springer, 1990; Vol. 95, p 71. (22) Sunday, M. E.; Hua, J.; Reyes, B.; Masui, H.; Torday, J. S. Anat. Eec. 1993, 236, 25. (23) Polverini, E.; Casadio, R.; Neyroz, P.; Masotti, L. Arch. Biochem. Biophys. 1998, 349, 225. (24) Coy, D. H.; Heinz-Erian, P.; Jiang, N.-Y.; Sasaki, Y.; Taylor, J.; Moureau, J.-P.; Wolfrey, W. T.; Gardner, J. D.; Jensen, R. T. J. Biol. Chem. 1988, 263, 5056. (25) Rivier, J. E.; Brown, M. R. Biochemistry 1978, 17, 1766. (26) Schwyzer, R. Biochemistry 1986, 25, 6335. (27) Spindel, E. R.; Giladi, E.; Segerson, T. P.; Nagalla, S. Recent Prog. Horm. Res. 1993, 48, 365. (28) Krane, I. M.; Naylor, S. L.; Helin-Davis, D.; Chin, W. W.; Spindel, E. R. J. Biol. Chem. 1988, 263, 13317.
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