rXXXX American Chemical Society A dx.doi.org/10.1021/jp1107153 | J. Phys. Chem. C XXXX, XXX, 000–000

ARTICLE

pubs.acs.org/JPCC

SERS, Molecular Dynamics and Molecular Orbital Studies of theMRKDV Peptide on Silver and Membrane SurfacesAlvaro E. Aliaga,† Hern�an Ahumada,§ Karen Sep�ulveda,§ Juan S. Gomez-Jeria,† Carlos Garrido,† BorisE. Weiss-L�opez,*,§ and Marcelo M. Campos-Vallette*,†

†Laboratory of Vibrational Spectroscopy and §Laboratory of Molecular Physical Chemistry, Faculty of Science, University of Chile, POBox 653 Santiago, Chile

1. INTRODUCTION

Surface enhanced Raman scattering SERS has been an activeresearch area with important applications ranging from surfacechemistry to biological chemistry and biomedical analysis. Bio-sensors are intrinsically associated with peptide-metal nanopar-ticle systems.1-3 The peptide-metal nanoparticle interactionalso has important consequences on the optical properties of thenanoparticle itself. SERS of a number of amino acids, peptides,and proteins, acquired with various SERS active substrates, hasbeen reported. Recent SERS results by Di Foggia et al.4 indicatethat in the case of four alternating polar/nonpolar peptidesderived from the self-assembling peptide EAK 16 (Ac-AEA-KAEAKAEAKAEAK-NH2), the interaction with a TiO surfacetakes place mainly through carboxylate groups, and the self-assembled structure of the peptide on the metal surface ispredominantly β-sheet.

Mitchell et al.5 have recently reported that the SERS metho-dology is a key factor related to the success of the application ofmetal nanoparticles in the appearance of strong SERS featuresfrom oligopeptides; they also used statistical analysis methods forthe peptide detection by SERS. Wei et al.6 obtained the SERSspectra of three cysteine (Cys)-containing aromatic peptides andpenetratin bound to nanoshell substrates. They concluded thatthe aromatic amino acid residues provide the dominant featuresin the SERS and Raman spectra. From SERS spectra of severalpeptides composed of different combinations of proline, trypto-phan, and tyrosine, Seballos et al.7 concluded that the bindingwith a silver surface occurs through both the carboxylic end andthe aromatic amino acid moieties.

Recent results on the applications of SERS to peptides havebeen published by Podstawka et al.,8 who compared the adsorp-tion behavior of bombesin (BN) (pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) and five BN-relatedpeptides in silver colloidal solution. The peptide metal interac-tion is mainly verified through the pyrrole ring of tryptophan andthe aromatic ring of the phenylalanine components. They alsoinfer a weak interaction through particular skeletal fragments ofthe peptidic chain. These results are slightly modified in the seriesof peptides. Differences in organization and orientation of thepeptides on the metal surface by changing the silver colloidalsurface to a silver electrode surface were inferred.

Infrared techniques such as PM, RAIRS, and ATR allowed oneto propose that the peptide L-glutathione (γ-Gln-Cys-Gly)interacts with Au through the cysteine thiol group in a gold-water interface and through the glycine carboxylate in a gold-ethanol interface.9 A reflection absorption infrared spectroscopic(RAIRS) study on Ag was performed by Itoh et al.10 forLangmuir-Blodgett films of the palmitoyl ornithine (PO) andpalmitoyl lysine (PL) peptides. The authors concluded a parti-cular organization of the peptides at the surface.

The interaction of peptides and oligopeptides with metalsurfaces can occur through at least two different mechanisms:(a) electrostatic interaction between charges in the oligopeptideand residual charges existing on the metal surface and (b)coordination complexes formed between very active groups,

Received: November 9, 2010Revised: December 29, 2010

ABSTRACT: The MRKDV peptide, structurally associatedwith an immunomodulatory protein, was studied using surfaceenhanced Raman scattering (SERS), molecular dynamics (MD)simulations, and quantum chemical calculations. The SERSspectrum of the MRKDV peptide adsorbed on the silver surfaceis dominated by signals coming from the guanidiniummoiety ofthe arginine amino acid (R). Guanidinium is the intrinsic probethat drives the orientation of the peptide onto the silver surface.Molecular mechanics and extended H€uckel calculations of amodel of MRKDV interacting with a silver surface support theexperimental results. MD calculations representing the evolution of the peptide toward a model membrane were also performed.The guanidinium moiety interacts with the phospholipidic membrane surface. A hydrophobic C-terminal modification favors thepeptide membrane affinity.

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such as-SH,-COO-, imidazol in hystidine (His),11 and indolein tryptophan (Trp),12 with surface metal atoms. In addition,intermolecular interactions between oligopeptides should beconsidered. In this sense, the hydrophobic interactions betweennonpolar amino acid residues in the oligopeptides also play animportant role in both the three-dimensional conformation andthe mechanism of adsorption on the metal surface.

The metal ion binding properties of two fluorescent analoguesof trichogin GA IV, which is a natural undecapeptide showingsignificant antimicrobial activity, were studied by circular dichro-ism, time-resolved optical spectroscopy, and molecular me-chanics calculations.13 Binding of Ca(II) and Gd(III) to thestudied peptide was shown to promote a structural transitionfrom highly helical conformations to folded structures character-ized by the formation of a loop that embedded the metal ion.Time-resolved fluorescence spectroscopy revealed that the pep-tide dynamics is also remarkably affected by ion binding: peptide-backbone motions slowed down to the microsecond time scale.Finally, molecular mechanics calculations emphasized the role ofthe central Gly5-Gly6 motif, which allowed for the twisting of thepeptide segment that gave rise to the formation of the bindingcavity.

Several bioactive peptides exert their biological function byinteracting with cellular membranes. Structural data and infor-mation about the distribution inside lipid bilayers becomeessential for a detailed understanding of their mechanism ofaction. A detailed biophysical characterization of the interactionof the cell-penetrating S413-PV peptide withmodelmembraneswas performed.14 It has been demonstrated that the interactionsare essentially of electrostatic type. As a consequence of thisinteraction with negatively charged model membranes, theS413-PV peptide becomes buried into the lipid bilayer, whichoccurs concomitantly with significant peptide conformationalchanges consistent with the formation of a helical structure.

Using surface plasmon resonance, the binding properties of anew class of immunomodulating peptides derived from thetransmembrane region of the T cell antigen receptor on modelmembranes were determined.15 The dibasic core peptide wasfound to bind both zwitterionic and anionicmodelmembranes aswell as to T cell membrane preparations. By contrast, switching

one or both of the basic residues to acidic residues led to acomplete loss of binding to model membranes. The position ofthe charged amino acids in the sequence, the number of hydro-phobic amino acids between the charged residues, and substitu-tion of one or both basic to neutral amino acids were found toaffect binding.

Molecular dynamics simulations were performed on thesequence-symmetric cyclic decapeptide antibiotic gramicidin S(GS), interacting with a hydrated dimyristoylphosphatidylcho-line (DMPC) bilayer; Results were compared with a controlsimulation of the system in the absence of GS.16 The area perlipid, lipid tail order parameters, and quadrupole spin-latticerelaxation times data of the control simulation are in agreementwith experiments. The GS has little effect on the membranedipole potential or water permeability.

Atomic force microscopy has been used to probe the organi-zation of lipid monolayers and bilayers.17 The authors exploredpeptide-membrane interactions, with emphasis on microbiallipopeptides and tilted peptides.

By means of a combined approach of fluorescence spectros-copy and molecular dynamics, B. Orioni et al.18 studied themechanism of membrane perturbation by the antimicrobialpeptide PMAP-23. Depth-dependent fluorescence quenchingexperiments and peptide-translocation assays were employedto determine the location of the peptide inside the membrane.Molecular dynamics (MD) simulations were performed startingfrom a randommixture of water, lipids, and peptide following thespontaneous self-assembly of the bilayer. Both experimental andtheoretical data indicated a peptide location just below the polarhead groups of the membrane, with an orientation essentiallyparallel to the bilayer plane. The atomic detail provided by thesimulations suggested the occurrence of an additional, morespecific, novel mechanism of bilayer destabilization by PMAP-23involving the unusual insertion of charged side chains into thehydrophobic core of the membrane.

The aim of the present contribution deals with the study bySERS spectroscopy of the N-terminal motif peptide, methiony-larginyllysyl-R-aspartylvaline MRKDV (see Figure 1), from thesubunit A of the immunogenic Concholepas concholepas hemo-cyanin.19 To complete the analysis of the SERS experiments, a

Figure 1. MRKDV-Ag final optimized geometry. Ball-and-stick molecular model displays the chemical element symbols. The silver surface appears ingray, and the peptide atoms are carbons, light blue; nitrogen, dark blue; oxygen, red; sulfur, yellow; and hydrogen, white.

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theoretical study based on the extendedH€uckel theory and 6-31G*methods for a simplified molecular model of the analyte-metalsurface interaction is proposed. Different experimental techniquesprovide evidence that supports the assumption that the role ofsubunit A, containing the motif MRKDV, seems to be crucial forthe proper immunomodulatory functioning of the protein invertebrates.20 With this perspective in mind, we extend the SERSstudywith aMD study of the behavior of the isolatedmotif peptidein a phospholipidmembrane environment.With this contribution,we expect to provide information such as peptide location,interactions with the membrane components, and modificationsof the MRKDV conformational structure during the possibleprocess of incorporation that might be essential for understandingthe participation of the motif peptide in the subunit-membraneinteraction. The MD simulation study is performed for MRKDVin a zwitterionic bilayer made of a mixture of dimyristoylpho-sphatidylcholine (DMPC) and dihexanoylphosphatidylcholine(DHPC) in water. To account for the possible hydrophobiceffects induced by the rest of the protein, a more hydrophobicderivative, the pentapeptide methionylarginylysyl-R-aspartyl-N1-(1,1 diethylpropyl)valineamide (MRKDVhd), was also stu-died. Targeting and understanding the biophysical interactionsbetween peptides and membranes at their site of action isparamount for the description of cell functioning and drug design.

2. EXPERIMENTAL SECTION

2.1. Sample. Synthetic water-soluble peptideMRKDVhighlypurified was purchased from Operon and used as supplied(g98.5% purity). Stock solutions of the peptide in water wereprepared to a final concentration of 10-4 M in nanopure water.2.2. Preparation of Silver Nanoparticles. Silver nanoparti-

cles (AgNPs) were prepared by chemical reduction of silvernitrate with hydroxylamine hydrochloride.21 The resulting col-loids display a final pH close to 7. The aqueous solutions used forthe AgNPs formation were prepared by using nanopure water.Colloids showed a milky gray color.2.3. Preparation of SERS Samples. Aliquots of the aqueous

peptide solution 10-4 M were added onto a quartz slide (0.5-1 μL) and dried at room temperature; a film of the dried peptidewas obtained. Then the Ag colloidal suspension was added(∼50 μL) onto the dried peptide sample to form peptide-NPaggregates. SERS measurements were performed at roomtemperature.2.4. Instrumentation. The Raman and SERS spectrum of the

peptide was measured with a Renishaw micro-Raman system(RM1000). The sample was photostable when probed with laserlines at 514, 633, and 785 nm. The best spectral data wereobtained by using the 633 nm laser line. This instrument wasequipped with a Leica microscope and an electrically refrigeratedCCD camera. The signal was calibrated by using the 520 cm-1

line of a Si wafer and a 50� objective. The laser power on thesample was 2 mW. The resolution was set to 4 cm-1, and 5-20scans of 10 s each were averaged. The spectral scanning condi-tions were chosen to avoid sample degradation.2.5. Spectral Reproducibility. No reproducible SERS spec-

tra were obtained by using the traditional way to prepare thesample; that is, by addition of the sample solution to the colloidalsuspension or the inverse. The SERS reproducible spectra frombatch to batch were obtained by adding the colloidal suspensiononto the dried analyte sample.

3. THEORETICAL CALCULATION

3.1. Molecular Models, Methods and Calculations. Tocomplete the analysis of the SERS experiments, a theoreticalstudy was performed. A simplified molecular model for theanalyte surface interaction is proposed. The silver atoms surfacewas the same employed in our previous studies.22-25 Briefly, aface centered cubic structure with a = 0.408 nm and 9 � 9 � 2cells was built and trimmed to get a planar double layercomposed of 324 silver atoms. Since the experimental conditionsof the SERS study, the MRKDV peptide was studied as aguanidinium form.Molecular mechanics was employed to optimize theMRKDV-

Ag geometry. The bilayer geometry was kept constant.The peptide was placed at different distances and orientationsfrom the center of the Ag bilayer. Figure 1 shows the finalgeometry.Extended H€uckel theory (EHT) was used to calculate the

wave function of MRKDV as an isolated system and interactingwith the metal surface. The Hyperchem program was used.26

EHT calculations produce qualitative or semiquantitative de-scriptions of molecular orbital and electronic properties.27 Thecombination of EHT with molecular mechanics was able to give,for example, a qualitative explanation of our previous SERSworks in nanotubes,22 humic acids,28 tryptophan,23 lysine,24 andarginine25 interacting with Ag surfaces.3.2. Molecular Dynamics Simulation. A bilayer made of 72

DMPC and 24 DHPC molecules surrounded by 7049 SPC29

water molecules was assembled in a box of dimensions 5.7 �5.3 � 10.3 nm3, with periodic boundary conditions in alldirections of space. This system was equilibrated for more than10 ns. After that, the simulation box was replicated three timeshaving a total of four simulations. One molecule of MRKDV wasincorporated in the hydrophobic core of the bilayer in simulation1, and one molecule of MRKDV, in the aqueous region ofsimulation 2. To study the effect of hydrophobic groups,presumably present in the original protein but not in MRKDV,the 3-ethylpentane-3-amine group was bonded directly to thecarbonyl atom of valine, forming a peptide bond, to obtain themore hydrophobic derivative MRKDVhd. Following the sameprocedure described before, one molecule of MRKDVhd wasintroduced in the hydrophobic region of simulation 3, and oneMRKDVhd, in the aqueous region of simulation 4. Figure 2shows a snapshot of simulation 2 after 5 ns of trajectory.Calculation of all trajectories and their further analyses weremade using the software package Gromacs v. 4.030 running in aSuper-Micro 4-blades cluster with 2 quad-core processors each.An attached atom representation was used for the protons in thealiphatic chains of the lipids. For trajectory visualization andgraphics, the VMD program31 was employed. Bonded andnonbonded interactions were calculated using the Gromosforce field.32 LINKS was used to restrict the bond lengths, andSETTLE, to constraint the geometry of water molecules.33,34 A 1nm cutoff was used for the Lennard-Jones and real spaceelectrostatic potentials. Long-range electrostatic interactionswere calculated using PME.35-37 The neighbor list was updatedevery 10 time steps. Temperature (T) and pressure (P) were keptconstant at 300 K (all species independently coupled) and 1 barusing a weak coupling algorithm,38 with time constants of 0.1 and1.0 ps for T and P, respectively. Trajectories of 6, 6, 18, and 50 nswere calculated for simulations 1, 2, 3, and 4 respectively, all witha time step size of 2 fs.

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4. RESULTS AND DISCUSSION

4.1. SERS Spectra. A unique and reproducible SERS spec-trum in colloidal solution for the peptide was not achieved. The

SERS spectrum of the peptide in solid, that is, the analyte coatedby the metal nanostructure, was successful. The spectral analysisof the SERS spectrum was performed on the basis of the existingRaman data and our previous Raman and SERS band assign-ments for most of the amino acid residues of the presentpeptide.23-25,38-41 Raman and SERS spectra of the peptide aredisplayed in Figure 3. Table 1 contains the most probableassignment expressed in terms of the amino acid symbol. Detailsof the corresponding normal modes are given by Stewart et al.39

The strongest band of MRKDV at 1060 cm-1 and the weak onesat 990 and 845 cm-1 are ascribed to the guanidinium fragment ofArg (R).25 Other very weak bands are assigned to the amino acids

Figure 3. (A) Raman and (B) SERS spectra of the oligopeptideMRKDV. SERS spectrum was obtained by adding the colloidal suspen-sion onto the dried analyte sample.

Table 1. SERS Frequencies (cm-1) of the MRKDV Peptideand the Most Probable Band Assignment

MRKDV assignmenta

1615 δNH

1559 δNH

1447 D, K, Vb

1353 D, K

1307 D, K

1275 D, K, V

1175 K

1060 R

990 R

927 M

845 R

726 M, K, V, D

685 M

636 D, K

410 δCCNaM, methionine; R, arginine; K, lysine; D, aspartic acid; and V, valine.bFor the specific normalmodes involved in the vibration, please see ref 39.

Figure 2. Snapshot of simulation 2 after 5 ns of trajectory. DMPC molecules appear in gray; DHPC, in black; and water oxygen and hydrogen are redand white, respectively. DMPC, DHPC, and water are displayed by the bond dynamic drawing method. MRKDV peptide is displayed in the van derWaals and bonds drawing method with carbons in light blue, oxygen in red, sulfur in yellow, and nitrogen in dark blue.

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Met (M), Lys (K), Asp (D), and Val (V) (see Figure 3 andTable 1). The presence of these very weak bands, mostly ascribedto different vibrations of the carboxylic and amino groups, CH2

deformations, and particularly the νCS of Met at 685 cm-1,40

indicates that the corresponding amino acid residues also interactwith themetal surface. This interaction should be very weak, eventhough with a SERS favorable orientation of the chemical groupsinvolved.42 To precise the knowledge of the physical andchemical characteristics of the interaction, it should be necessaryto study the nature of the peptide metal interface.The Asp bands of MKRDV at 1447, 1307, and 726 cm-1

suggest that the Arg residue anchors the MRKDV peptide to thesurface through its guanidine group. This proposition agrees withthe results of Di Costanzo et al.,43 who described the stereo-chemistry of the guanidine-metal interactions in small mole-cules and proteins.4.2. MRKDV Metal Interaction. In the final MRKDV-Ag

geometry (see Figure 1), the sulfur atom is located at a distance of3.0-3.4 Å� from some Ag atoms. One of the N atoms of theguanidinium group is at 2.8-3.2 Å from the metal surface, andtheir attached H atoms is at distances of ∼2.40 Å from the Agsurface. The N amino and its H-bonded atoms are located at∼2.4 Å� from the silver surface. Two CO groups are in the range2.4-2.8 Å� away from the surface.The CO groups are placed at the center of the Ag surface area

in which the electronic density of the HOMO is zero. Theguanidinium moiety and the sulfur atom are also placed on partsof the metallic surface in which the electron density of the

HOMO is zero. This geometrical situation is the optimal one dueto the fact that the interaction of the oxygen lone pairs with theHOMO of the Ag surface must be minimal.As expected from the analysis of the isolated systems, the

results for theMRKDV-Ag system indicate that, as in the case ofzwitterion arginine, a charge transfer occurs from the valenceband of the Ag surface to the pentapeptide. The charge transfer isverified from the valence band of the Ag surface to the emptyLUMOs of the guanidinium moiety, the CO oxygen atoms andthe sulfur atom closest to the Ag surface.4.3. Molecular Dynamics. Figure 4 shows the center of mass

(c.o.m.) Z-coordinate of the complete peptides as well as the c.o.m. Z-coordinate for each amino acid component as a function oftime. Figures 4A-D corresponds to simulations 1-4, respec-tively. All these figures represent the upper interface of thesimulation box (see Figure 2). Three different regions, charac-teristic of all amphiphilic bilayers, can be distinguished: region Icorresponds to the aqueous phase, region II is the interface, andregion III is the hydrophobic core; that is, the center of the bilayer.The horizontal lines represent the limits of the interface, estimatedaccording to a previous definition.44 The width of the interface inthe absence of guest peptides is 1.1 nm, and the inclusion ofMRKDV decreases this value to 0.8-0.9 nm. The inclusion ofMRKDVhd has no significant effect on the width of the interface.As mentioned before, in simulation 1 (Figure 4A), MRKDV

was originally positioned in region III and remained there for therest of the simulation. It can be noticed that Val and Lys arepositioned closer to the interface, near the aqueous region.

Figure 4. Time evolution of the center of mass (c.o.m.) Z-coordinate of all amino acid components of MRKDV (A and B) andMRKDVhd (C and D).Panels A, B, C, and D correspond to simulations 1, 2, 3, and 4, respectively. The limits of the top interface are denoted by horizontal lines.

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However, when MRKDV is initially positioned in the aqueousregion (Figure 4B), a significant displacement toward the inter-face and modifications in the distribution of amino acids alongthe Z-coordinate are observed. The final distribution shows thatVal and Arg remained near the aqueous phase, whereas Met, Lys,and Asp become more incorporated into the interface. In thesefirst two simulations, Val contains the complete carboxylatemoiety and is always located near the aqueous region of thebox. The amine terminal of Met forms a strong hydrogen bondwith water at the upper limit of the interface in simulation 2 orwith water at the lower limit interface in simulation 1 (the lowerinterface limits are not indicated in Figure 4).When MRKDVhd is initially positioned in region III, it shows

a moderate displacement toward the interface (Figure 4C),without significant amino acids reordering; however, ifMRKDVhd is originally placed in region I near the interface(Figure 4D), it experiences a large displacement toward theinterior of the interface and a significant modification in thedistribution of amino acids along the Z-axis of the box. Metcrosses most amino acids to finally reach the lower interface tobecome hydrogen bonded to water. Arg is located at the interiorof the interface, possibly with the guanidine moiety interactingwith the phosphate groups. In the last two simulations, Val isdirectly attached to the hydrophobic substituent, and as ex-pected, it locates closer to region III. The existence of crossingsamong the amino acids strongly suggests the occurrence ofconformational changes in the peptide. This is discussed in moredetail later.

To examine the distribution of the amino acids along the Z-axis of the box, mass density profiles of the equilibrated systemswere calculated for all simulations. The results are displayed inFigure 5A-D for simulations 1-4, respectively. It can beobserved that when either MRKDV or MRKDVhd is initiallylocated in region III (Figure 5A and C), all amino acids appeardistributed over this entire region, without significant crossing toregion II; however, the distributions of MRKDVhd are narrowerthan the distributions of MRKDV. This is clearly observed forLys and Met. If MRKDV or MRKDVhd is initially located inregion I (Figure 5B and D), the distributions of MRKDV are alsowider than the distributions of MRKDVhd. Furthermore, theresidues of MRKDV appear distributed around the interfacebetween regions I and II, whereas the residues of MRKDVhd aredistributed around the interface between regions II and III,deeper into the hydrophobic core. This is expected for the morehydrophobic derivative.The results from the simulations show that neither MRKDV

nor MRKDVhd is able to cross the interface and penetrate thehydrophobic core of the membrane. When they are initiallypositioned in the aqueous phase, region I, they spontaneouslydisplace toward the interior of the bilayer, with the finalequilibrium location being a function of the hydrophobic char-acter of the peptide. However, when originally located in thehydrophobic core, region III, they remain there.To test for a possible interaction between the guanidine

moiety of Arg and the phosphate groups from the phospholipids,the radial distribution function of phosphate groups fromDMPC

Figure 5. Mass density profiles of all amino acid components ofMRKDV (A and B) andMRKDVhd (C andD) along theZ-axis of the box. Parts A, B, C,and D correspond to simulations 1, 2, 3 and 4, respectively. The limits between regions I, II, and III are denoted by vertical lines.

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around the guanidine moiety of Arg was calculated from simula-tion 3, and the result is shown in Figure 6. The first maximum canbe estimated at a distance of 0.6 nm and corresponds to twophosphate groups surrounding the guanidine fragment. Theexistence of a specific electrostatic interaction between theguanidine moiety and the phosphate groups fromDMPC cannotbe discarded. Even more, a strong and persistent hydrogen bondbetween the guanidine group and the phosphate head groupsfrom DMPC is detected.The observed crossings among the amino acids along the Z-

coordinate, suggest the existence of conformational changeswhen the environment of the peptide is modified. To detectpossible conformational changes of the peptides when located indifferent regions of the simulation box, we have calculated theaverage values of the three principal axis inertia moments ofMRKDV and MRKDVhd as a function of time in all simulations.An oblate symmetric body possesses two identical moments ofinertia, which are smaller than the unique inertia moment,colinear with the symmetry axis of the oblate. A prolate possessestwo equal inertia moments that are greater than the uniqueinertia moment, which is along the symmetry axis of the prolate.A nonsymmetric body has all inertia moments different. Calcu-lated average values of the principal axis inertia moments aredisplayed in Table 2. When MRKDV approached the interfacefrom either region III or I (simulations 1 and 2), the startingstructure was nonsymmetric in both cases. Along the simulations,the peptide becomes a prolate.On the other hand, when MRKDVhd approaches region II

from regions III and I (simulations 3 and 4), in both cases, it startsas a prolate symmetric body. Along simulation 3, no significantmodifications to the structure were observed and it remains aprolate; however, along simulation 4, the original prolate struc-ture becomes a nonsymmetric body. One of the energy compo-nents that affects the three-dimensional structure of proteins andpeptides is hydrogen bonds. To explore the origin of the forcesresponsible for the different conformation, the existence ofintramolecular hydrogen bonds between carboxylates and am-monium groups was tested. When MRKDVhd is located nearregion III, two strong hydrogen bonds were detected betweenAsp and Arg and between Val and Lys. When it is closer to regionII, it becomes a nonsymmetric body with no significant

intramolecular hydrogen bond. All the above results are consis-tent with an important conformational change of MRKDVhdwhen going from region I to II, with a final average structure thathas no symmetry. However, when MRKDVhd goes from regionIII to II, it remains as a prolate structure, stabilized by intramo-lecular hydrogen bonds.

5. CONCLUSIONS

The peptide MRKDV, structurally associated with an immu-nomodulatory protein, was vibrationally studied by using surfaceenhanced Raman scattering SERS and both quantum mechanicsand molecular dynamics approaches. No sample degradation(photo or thermal) was observed using 514, 633, and 785 nmlaser lines. The sample for SERS was prepared by adding the Agcolloid suspension onto the dried peptide sample, the wholedeposited on quartz slide. The MRKDV SERS spectrum isdominated by signals coming from the guanidinium moiety ofthe arginine amino acid (R); guanidinium is the intrinsic probethat drives the orientation of the peptide on the Ag surface.Theoretical calculations performed by using molecular me-chanics and extended H€uckel theory methods for a model ofthe MRKDV peptide interacting with a Ag cluster support theobserved experimental result. We also conclude that whenMRKDV, or the N1-(1,1-diethylpropyl)valineamide-substitutedpeptide (MRKDVhd) is initially located in the aqueous region, itspontaneously displaces toward the interior of the bilayer. Thefinal equilibrium position in the membrane is a function of thehydrophobic character of the peptide. When MRKDV becomesincorporated into the interface, it carries a significant amount ofwater from the interior to that region, decreasing the width ofregion II. When the peptides are initially positioned in region III,the center of the bilayer, they remain there. Depending on theenvironment, the peptide can display different conformations. Apersistent H-bond between the guanidine moiety of arginine andthe phosphate groups from DMPC was detected. Finally, theamine terminal from methionine forms a strong hydrogen bondwith water in either of the interfaces.

’AUTHOR INFORMATION

Corresponding Author*E-mail: (M.M.C.-V.) facien05@uchile.cl, (B.E.W.-L.) bweiss@uchile.cl.

’ACKNOWLEDGMENT

This contribution is financially supported by Fondecyt, Grants1090074 and 1095175. A.E.A. acknowledges AT 24090050 andDoctoral Fellowship CONICYT.

Figure 6. Radial distribution function of the phosphate groups sur-rounding the guanidine moiety from arginine. The first maximum at 0.6nm corresponds to two phosphate groups. The origin is located at thecenter of mass of the guanidine moiety.

Table 2. Average Principal Axis Moments of Inertia ofMRKDV and MRKDVhd, (a.m.u. � nm2) When Located inDifferent Environments: The Interface (region II) and Closerto the Hydrophobic Core (region III)a

Ixx Iyy Izz

region II MRKDVhd 110( 5 149( 11 184( 10

region III 217( 19 255( 19 74( 6

region II MRKDV 161( 24 192( 19 72( 10

region III 130( 10 153 ( 11 44( 4a Errors correspond to standard deviations.

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