Bioelectrochemistry 87 (2012) 21–27

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Bioelectrochemistry

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Peptide molecular junctions: Distance dependent electron transmissionthrough oligoprolines

Joanna Juhaniewicz, Slawomir Sek ⁎Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland

⁎ Corresponding author. Tel.: +48 22 8220211x244;E-mail address: slasek@chem.uw.edu.pl (S. Sek).

1567-5394/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.bioelechem.2011.11.013

a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 June 2011Received in revised form 13 November 2011Accepted 28 November 2011Available online 7 December 2011

Keywords:Electron transferMonolayerMolecular junctionScanning tunneling spectroscopyPeptide

Wehave investigated the efficiency of electron transmission through thiolated oligoproline derivatives of gener-al formula: Cys–(Pro)n–CSA, where CSA is a cystamine linker and n=1–4. The conductancemeasurements wereperformed using STM-based molecular junction approach.We have noted that the conductance of the oligopro-lines decays exponentially with increasing length of the molecules and the decay constant was 4.3 nm−1. Thisindicates that electron transfer is dominated by superexchange mechanism. Based on this observation, wehave concluded that the height of the barrier is affected by the specific conformation of the peptide backbone.Such conclusion is supported by the fact that the oligoprolines do not form intramolecular hydrogen bonds,which could provide alternative electron transfer pathways.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Electron transfer properties of the peptides attract much attentiondue to their important role in fundamental biological processes [1–3].Recently, peptides are often considered as building blocks for thedesign of functional bionanomaterials, biosensors, nanodevices ormatrices for immobilization of biological material [4–10]. It is knownthat peptides can act asmediating bridges for long range electron trans-fer (e.g. in proteins) and the efficiency of this processmay be affected bythe details of the primary and secondary structure. This opens thepossibility of fine-tuning the conductance of the molecule by carefulselection of the amino acid sequence and the conformation of the pep-tide. However, the reliable investigations of peptide-mediated electrontransfer involve consideration of number of factors like the length of themolecule, the sequence of amino acids, molecular dipole moment,presence of complex ions, the secondary structure and presence ofhydrogen bonds. The influence of the secondary structure of peptideon electron transfer efficiency was demonstrated by several groups[11–20]]. It was found that the peptides with α-helical and 310- helicalconformation are good mediators for long range electron transport[11–17]. Similar results were obtained for collagen-like structures[18]. Quite often, the hydrogen bonds were considered as one of themost important factors enhancing electron transmission throughpeptides with well-defined secondary structure [21–23]. The hydrogenbonds, either intramolecular or intermolecular, are expected to createadditional pathways for electron transport and contribute to the

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lowering of the barrier for electron transmission [21–23]. As a conse-quence, the peptides exhibiting the secondary structure with hydrogenbonded network should promote electron transfer more efficientlycomparing to non-hydrogen bonded systems like polyprolines. In thispaper we would like to demonstrate whether such assumption isreasonable or not.

Formation of specific secondary structure in peptides is determinedby the sequence of individual amino acids. As an example, it is well-known that the presence of alanine and aminoisobutyric acid in thepeptide chain highly promotes formation of α-helical structure, whilevaline or phenylalanine contribute to formation of β-sheet structure[24]. However, proline exhibits unique properties among otherstandard amino acids. It contains a cyclic backbone with a secondaryα-amino group, therefore, it is not able to form intramolecular hydro-gen bonds. Oligoproline chains composed of at least three proline resi-dues adopt two different, well-defined secondary structures —

polyproline I and II [25]. Poliproline I (PPI) is a dense, right-handedhelix with 3.3 residues per turn and 0.19 nm rise per residue and allamide bonds are in cis conformation (ω=0°). Poliproline II (PPII) isrelatively open, left-handed helix with 3 residues per turn and 0.31 nmrise per residue and the peptide bonds adopt trans conformation(ω=180°). Due to the aforementioned structural features, prolineexhibits a specific stereochemical behavior and the proline oligomersare often used as amolecular ruler [26] or scaffold [27]. Moreover, oligo-prolines are believed to form quite rigid backbones, therefore, they havebeen frequently used to study the distance dependence of electrontransfer in peptides using photochemical [28–32], electrochemical [33]and pulse radiolysis [34,35] methods.

In this paper, we report the conductance measurements of singleoligoproline-based peptide molecules with different length. The

22 J. Juhaniewicz, S. Sek / Bioelectrochemistry 87 (2012) 21–27

efficiency of electron transfer through these peptides was investigat-ed using STM-based molecular junction method involving the entrap-ment of single molecules between gold substrate and gold STM tip. Sofar, this approach was used for the conductance measurements ofalkanedithiols, DNA, peptides, diamines, dicarboxylic derivatives, vio-logens and diisonitryles [36–42]. In this study, the junctions wereformed using self-assembled monolayers of peptides of formula(Boc) Cys(Acm)–(Pro)n–CSA, n=1–4 (See Scheme 1). The structureof peptides was examined using circular dichroism spectroscopy. Byincreasing the number of proline residues in peptide chain, it waspossible to investigate the efficiency of electron transfer through pep-tide molecules by determining the decay constant. The effect of theformation of the secondary structure on the effectiveness of mediatedelectron transmission was also considered.

2. Materials and methods

All chemicals were purchased from Aldrich, Fluka and POCh (Gli-wice, Poland) and used without further purification.

2.1. Synthesis of (Boc) Cys (Acm)–(Pro)1–4–CSA

(1) A mixture of 5.5 mmol of Boc–Pro–OH, 8.0 mmol of HOBt and8.0 mmol of TBTU in 50 ml of dichloromethane was stirred for 30 minat 0 °C in an ice bath and then the solution of 2.75 mmol of cystaminehydrochloride and 5.5 mmol of triethylamine in 20 ml of dichloro-methane was added. The resulting mixture was stirred for 12 h andthen extracted with 50 ml portions of saturated aqueous solution ofNaHCO3, 10% citric acid, saturated NaHCO3 and water. The organicphase was dried over anhydrous MgSO4 and filtered. After that, solventwas removed using rotary evaporator. The resulting solid was treatedwith 10 ml of trifluoroacetic acid for 30 min in 0 °C in an ice bath.After this time, the solvent was evaporated under vacuum and a mix-ture of 5.5 mmol of triethylamine and 50 ml of dichloromethane wasadded. The solution was left for 30 min in ambient conditions. Aboveprocedure was repeated two, three and four times for CP2CSA, CP3CSAand CP4CSA, respectively. (2) The solution of 5.5 mmol of Boc–Cys(Acm), 8.0 mmol of HOBt and 8.0 mmol of TBTU in 50 ml of dichloro-methane was stirred for 30 min at 0 °C in an ice bath. Then, it wasmixed with the solution obtained from step (1), and the resulting mix-ture was further stirred for 12 h. Next, the reaction mixture wasextracted with 50 ml portions of saturated aqueous solution ofNaHCO3, 10% citric acid, saturated NaHCO3 and water. The organicphase was dried over anhydrous MgSO4, filtered and then the solventwas removed under vacuum. The crude product was purified usingflash column chromatography on silica gel. ESI-MS: CP1CSA m/z=

Scheme 1. Chemical structures of the oligoproline-based peptides.

917.7 [M+Na]+, Mcalc=895.2 g/mol; CP2CSA m/z=1111.8 [M+Na]+,Mcalc=1088.5 g/mol; CP3CSA m/z=1306.9 [M+Na]+, Mcalc=1283.6 g/mol; CP4CSA m/z=1499.7 [M+Na]+, Mcalc=1476.7 g/mol.It should be noted that all synthesized oligoprolines are symmetricdisulfides (as shown in Scheme 1), however, after adsorption on goldthey form monolayers indistinguishable from those obtained usingthiol derivatives [43].

2.2. Monolayer preparation

Gold substrates (Arrandee™, Werther, Germany) were flameannealed before use in order to obtain flat Au (111) terraces. Theadsorption of peptides was carried out by self-assembly from 1 mMethanolic solutions for 24 h. Then, the sampleswere rinsedwith ethanoland water and dried in an Ar stream. In the next step, the substratesmodified with peptides were subjected to Acm deprotection procedureand subsequently used to formmolecular junctions. In order to removeAcmprotecting group, the peptide-modified electrodeswere immersedin deoxygenated water and then the pH was adjusted to 4.0 using am-monia and acetic acid. The resulting solution was bubbled with argon.Next, mercury (II) acetate was added and the resulting solution wasbubbled with argon for 45 minutes. Subsequently, β-mercaptoethanolwas dropped in and after 15 minutes the substrates were removedfrom the solution, washed with water and dried in an argon stream.After that, the samples were ready to form the junctions with the topcovalent Au–S contact. It is necessary to note that during all measure-ments the amino group in cysteine residue remained protected withBoc moiety in order to prevent formation of alternative Au–N contact[44].

2.3. Electrochemistry

The cyclic voltammetry (CV) and electrochemical impedance spec-troscopy (EIS) experiments were performed in a three-electrode cellwith peptide-modified gold electrode, platinum foil and Ag/AgCl (sat.KCl) serving as working, counter and reference electrode respectively.The supporting electrolyte was either aqueous 0.1 M KNO3 (for EISmeasurements) or aqueous 0.5 M KOH (for CV desorption experi-ments). The oxygenwas removed from the solution using an Ar stream.All electrochemical measurements were performed using CHI 750Bbipotentiostat (CH Instruments Inc., Austin, TX). All data were recordedat room temperature.

2.4. Circular dichroism

Circular dichroism (CD) spectroscopy experiments were performedusing J-815 CD Spectrometer (JASCO Inc., Easton, MD). A quartz cuvetteof 1 cm length was used as the optical window. All CD spectra wererecorded at room temperature. The solvent was ethanol and theconcentration of peptides was kept at 0.1 mM.

2.5. Quartz microbalance

For the quartz crystal microbalance (QCM) measurements the PC-controlled microbalance (Eureka, BIOAGE s.r.l., Italy) with 6 MHz goldcoated AT-cut quartz crystals were used. The geometrical surface areaof the gold electrode was 0.21 cm2 and the real surface area wasdetermined before each experiment by a surface oxide formationprocedure in 1 M H2SO4. The viscosity and density of the solutionwere kept constant during the experiments and the mass changewas calculated from the change of frequency according to Sauerbreyequation [45].

23J. Juhaniewicz, S. Sek / Bioelectrochemistry 87 (2012) 21–27

2.6. Formation of molecular junctions

Scanning tunneling spectroscopy (STS) experiments were per-formed using MultiMode SPM instrument working with NanoscopeIIIa controller (Veeco Instruments Inc.). The data were recorded underambient conditions in air. Mechanically cut 0.25 mm gold tips(99.99%, Aldrich) were used in the conductance measurements. Molec-ular junctions were formed using STS method based on the measure-ments of the current as a function of the distance (z). The junctionswere formed by placing the gold STM tip was at given location on thepeptide-modified surface and the initial distance between the tip andthe sample was determined by bias voltage and tunneling current set-tings. The setpoint values were in the range of 0.2–9 nA depending onthe monolayer thickness. We found that these settings were sufficientto bring the tip into the contact with the molecules adsorbed on thegold electrode.When the proper distance between the tip and the sam-plewas achieved, the tipwas expected to interact with free thiol groupsof themolecules anchored on the gold electrode and the chemical Au–Scovalent bonds were formed. In the case of I (z) spectroscopy, the feed-back was disabled and the tip was lifted at the rate of 16 nm/s whilekeeping constant x–y position and the current was recorded as a func-tion of the tip – substrate distance. The conductance of the junctionswas measured at different bias voltages within the range of ±500 mV.In order to obtain a reliable statistics, the measurement was repeated200–300 times for each sample at each bias. Five independent samplesfor each peptide were studied.

3. Results and discussion

3.1. Characteristics of the monolayers

As it was already mentioned, oligoproline formed of at least threeproline residues can exist in two different helical conformations:polyproline I (PPI) and polyproline II (PPII), which differ in length.Therefore, it was crucial to verify which structure dominates for oli-goprolines studied in this work. PPI and PPII helical structures canbe distinguished by circular dichrosim (CD) spectroscopy [46]. TheCD spectrum of PPI exhibits a medium negative band at 200 nm, astrong positive band at 215 nm and a weak negative band at 232 nm,while the PPII spectrum exhibits a strong negative band at 206 nmand a weak positive band at 228 nm [25,47]. Fig. 1 presents CD spectrarecorded in the solutions of the peptide molecules studied here. Allcompounds exhibit a strong negative band at 204–206 nm and a weakpositive band at 222–230 nm. Thus we concluded that peptide bondsin CPnCSA, where n=1–4, preferentially adopt trans conformation.However, formation of the secondary structure involves at least 3amino acids residues, therefore, well-defined rigid PPII structure can

Fig. 1. CD spectra recorded for CPnCSA where n=1–4. Solvent: ethanol; peptides con-centrations: 0.1 mM.

be observed for CP3CSA and CP4CSA, while CP1CSA and CP2CSA areslightly more flexible. Based on CD data, we have calculated the dis-tances between terminal sulfur atoms for oligoprolines investigatedhere using following dihedral angles φ=−750 ψ=1450; ω=1800

[24]. The distances (ds-s) were 1.11 nm, 1.42 nm, 1.73 nm and 2.04 nmfor CP1CSA, CP2CSA, CP3CSA and CP4CSA respectively.We have assumedthat the conformation of the peptides remains the same after adsorp-tion of peptides on gold and these numberswere further used for deter-mination of distance dependence for electron transmission througholigoprolines trapped within the junction.

The quality of the monolayers formed by Acm-protected peptideswas examined by cyclic voltammetry and electrochemical impedancemeasurements. Fig. 2 presents the examples of the cyclic voltammetriccurves recorded for gold electrodes modified with peptide monolayers.When the potential of the peptide modified electrode is swept to nega-tive value, a cathodic peak is observed, which corresponds to the reduc-tive desorption of the molecules from gold surface. During the reversescan, re-adsorption of themolecules occurs, which is reflected by an an-odic peak. Such shape of voltammetric curves is typical for desorption/adsorption process of thiolated compounds indicating that the peptidemolecules are chemisorbed on the gold electrode [48]. As shown inFig. 2 desorption potentials shift to the less negative values withincreasing number of proline residues in peptide chain (see alsoTable 1). The position of desorption peak provides an informationabout the intermolecular interaction within the monolayer [48]. Thefilm becomes less compact, as the interactions between adjacentmolecules become less attractive. As a result, less negative potential isneeded to remove the molecules from the electrode. Therefore, thedifferent desorption potentials of the peptides can be attributed todifferent packing densities. If we consider this, the packing density ofoligoproline monolayers decreases with increasing number of prolineresidues. However, desorption of CP3CSA and CP4CSA occurs at veryclose potentials, which suggests that the difference in monolayer pack-ing is quite small for this pair. It should be noted that the results ofcapacitance measurements lead to exactly the same conclusions (seeTable 1). The values of the capacitance measured for modified elec-trodes reflect the permeability of the monolayer for ions and solventmolecules. More permeable monolayer gives higher value of the capac-itance since it is less densely packed. If we consider the results collectedin Table 1, it is clear that the permeability of the oligoproline mono-layers increases with increasing number of proline residues. In otherwords, longer molecules produce less densely packed monolayer.However, again the packing densities of CP3CSA and CP4CSA mono-layers seem to be similar since they exhibit quite alike permeabilityfor ions and solvent molecules. All these observations are fullyconfirmed by the results obtained from QCM experiments, where thesurface concentrations for the CPnCSA monolayers were determined.

Fig. 2. Cyclic voltammetry curves recorded for gold electrodes modified with self-assembled monolayers of CPnCSA, where n=1–4. Supporting electrolyte: 0.5 M KOH.

Table 1Characteristics of peptide monolayers.

Monolayer Peak potentialfor reductivedesorption/ V

CapacitancefromEIS/ μF×cm−2

QCM measurements

Г/ mol×cm−2 Molecularareaa/ nm2

CP1CSA −1.11±0.01 6.93±0.50 (4.2±0.1)×10−10 0.39±0.01CP2CSA −1.01±0.01 7.33±0.68 (3.5±0.1)×10−10 0.48±0.01CP3CSA −0.94±0.02 9.11±0.79 (2.6±0.1)×10−10 0.64±0.02CP4CSA −0.93±0.01 10.00±0.97 (2.5±0.1)×10−10 0.66±0.02Bare Au — 19.0±1.0 — —

aper single peptide chain adsorbed on gold surface.

Fig. 3. Representative current–distance curves obtained in individual junction experi-ments with (A) CP1CSA, (B) CP3CSA. The grey dashed curves were recorded usingbare gold substrates. The insets show the histograms constructed on the basis ofcurrent–distance curves recorded for peptides. Bias voltage=+0.5 V.

Fig. 4. Logarithmic plot of the current as a function of the peptide length defined as adistance between terminal sulfur atoms (ds-s). The value of decay constant (β) wasobtained from the slope of this plot (see Eq. (1) in the text). Currents were determinedfrom I (z) curves recorded at bias voltage of +0.5 V.

24 J. Juhaniewicz, S. Sek / Bioelectrochemistry 87 (2012) 21–27

As shown in Table 1, the area occupied by single peptide chain ongold surface depends on the length of the oligoproline backboneand the packing density decreases in the sequence CP1CSA>CP2CSA>CP3CSA≈CP4CSA. Clearly, longer peptide chains require more space.Therefore, the monolayers become less compact and more permeablefor hydrated ions and solvent molecules. Such behavior is connectedwith the fact that CP3CSA and CP4CSA are long enough to form relativelyrigid PPII helical structure. Therefore, they differ only in length, whilethe spatial requirements and resulting packing density and the perme-ability observed for the monolayers formed by these molecules remainunchanged. Shorter analogs (i.e. CP1CSA and CP2CSA) are more flexibleand their backbones can adoptmore extended formswhich require lessspace. This leads to formation of the monolayers with higher packingdensity and lower permeability for ions and solvent.

3.2. Electron transmission through oligoprolines

Prior to the junction formation, the peptide-modified substrateswere subjected to Acm deprotection procedure in order to obtain freethiol groups in an external plane of themonolayer. This step is essentialfor further formation of the Au–S bond between the STM tip and themolecules immobilized on gold substrate. The conductance measure-ments were carried out for the bias voltages ranging from −0,5 V to+0,5 V. Fig. 3 presents exemplary current-distance curves recorded injunction experiments using bare gold (gray dashed curves) and the sub-strates modified with CP1CSA and CP3CSA ((black solid curves). Eachcurve was obtained in a single junction experiment. For bare gold, afast exponential decay of the current with increasing distance betweenthe tip and the gold electrode is observed, that is typical for tunnelingthrough the empty gap. The curves recorded for peptide-modifiedsubstrates present substantially different characteristics. Initially, thecurrent decays exponentially with increasing distance between theelectrode and the STM tip. Then the current plateau appears, althoughthe absolute distance between tip and sample still increases. This isrelated to the fact, that the electron transfer occurs through the “path-way” constituted by the molecule or molecules bonded between thetip and the sample. Thus the electron transfer distance remainsunchanged as long as the molecule is bonded to the metallic contacts,therefore, the current is nearly constant and it can be ascribed to theconductance through molecule(s) immobilized within the junction.However, when the contact is broken, the conductance of the gapdecreases dramatically and the current drops to very low values,below detection limit. The insets in Fig. 3 show histograms obtainedon basis of many current-distance curves. In case of all peptides wehave observed pronounced peaks corresponding to the conductancethrough one or two peptide molecules trapped between metallic con-tacts. For further analysis, we used the current values ascribed to thefirst peak on the histogram, i.e. corresponding to current flow througha single molecule. Comparing the histograms obtained for oligoprolinesat given bias, we have noted that the conductance decreases in asequence: CP1CSA>CP2CSA>CP3CSA>CP4CSA. This is illustrated inFig. 4, which shows that the current (I) decays exponentially with

increasing length (ds-s) of the peptide backbone accordingly to the fol-lowing equation:

I ¼ I0exp −βds−sð Þ ð1Þ

where I0 is the current at ds-s=0 and β is a decay constant which char-acterize the efficiency of electron transmission through the junction.Such distance dependence is indicative for superexchange mechanismof electron transfer. This observation is in agreementwith experimental

25J. Juhaniewicz, S. Sek / Bioelectrochemistry 87 (2012) 21–27

and theoretical results [29,49]. The decay constant (β) determined fromthe slope of the logarithmic plot presented in Fig. 4 is 4.5±0.2 nm−1.However, the contact conductance (i.e. at ds-s=0), which can be deter-mined from this plot is several orders of magnitude lower than contactconductance values for Au–S bond reported by Tao and other groups[39,50]. This is quite surprising, however, even lower values of contactconductancewere observed for other systems displaying low decay fac-tors, for example for carotenoids and porphyrin oligomers [51,52]. Theorigin of such behavior is unclear since numerous factors can influencethe contact conductance. For example the conductance may be affectedby oxidation of thiolates to sulfonates as shown by Jang and Goddard[53]. Also the changes in the geometry of the contact may cause largevariation in the conductance [54]. Bautista and coworkers have shownthat the electrical behavior of the peptides depends on the number ofmetal atoms directly contacting the molecule within the junction [55].Moreover, the value of the decay factor may change for very short brid-ges [56]. Since it is difficult to point out the single origin of low contactconductance, we decided to limit our analysis of the electron transportbehavior to the comparison of the decay factor. The latter depends onintrinsic bridge energy and is not related to metal-molecule contacts.Thus comparing the decay constants we can conclude about the abilityof the given bridge to promote electron transfer. Such approach isreasonable if we consider that even for the same bridges different con-ductance values are reported, e.g. for alkanedithiols two sets of conduc-tance reflecting different the geometry of Au–S bond were reported byTao [39]. In the case of oligoprolines we have observed only one set ofconductance. We believe it results from the experimental approachwe have applied. The junctions were formed using pre-assembledmonolayers. Thus the sulfur atoms occupy energetically favorable siteson gold substrate, most likely three-fold hollow sites [57]. It meansthat the geometry of the bottom contact (i.e. between the moleculeand the gold substrate) is probably the same for all molecules. More-over, it does not vary during the junction formation since the tip pene-trates the monolayer but it does not hit the substrate as in breakjunction method.

Fig. 5 illustrates the dependence of the decay factor on bias voltageapplied between two metallic contacts. It changes slightly around thevalue of 4.3 nm−1. Such weak dependence indicates that the electronictunneling barrier is actually independent of the bias voltage. Similarobservations were reported for alkanethiols trapped between twomercury electrodes and it was concluded that the electron tunnelingthrough alkanethiolates does not conform classical square barriermodel [58]. Such behavior was identified as a signature of thethrough-bond mode of the electron tunneling. Our results indicatethat oligoprolines show similar characteristics. The average decayconstant of 4.3 nm−1 obtained in present work suggests quite goodelectron mediating properties of oligoproline bridges, comparable to

Fig. 5. The dependence of the decay constant (β) on the bias voltage obtained for oli-goproline junctions. The data was obtained on the basis of I (z) curves recorded at dif-ferent bias voltages within the range of±0.5 V.

α-helical peptides where the decay factor obtained from STM-basedjunction experiments was 5.0 nm−1 [37]. This is quite surprising sincein many cases, especially for helical structures, the increased ability ofthe peptide molecules to mediate electron transfer is ascribed to thepresence of intramolecular hydrogen bonds. It is believed that hydrogenbonds offer alternative pathways for electron transport and can lowerthe height of the tunneling barrier [21–23]. However, in the case ofoligoprolines the efficiency of electron transmission is comparable toα-helix, although the peptides adopt PPII structurewithout intramolec-ular hydrogen bonds. Then the question arises whether the hydrogenbonds are important for electron transfer or not. We believe they are,however, the role of the hydrogen bonds in mediating electron transferis complex, i.e. it is not limited to providing alternative electron transferpathways. Apparently, the ability of the hydrogen bonds to act as astructural motif which supports particular structure of the peptide isof crucial importance. It means that the efficiency of electron transferis strongly affected by the conformation of the peptide bridge (i.e. dihe-dral angles) and the intramolecular hydrogen bonds simply help tomaintain optimal structure of the backbone. Thus their ability toprovide additional electron transfer pathways seems to be an extraprofit. Such conclusion is supported by the results obtained in thiswork. The oligoproline backbone is relatively stiff due to the presenceof the pyrrolidine rings. Therefore, the structure of the oligoprolines iswell defined and provides efficient pathway for electron tunnelingalthough there are no hydrogen bonds.

Another interesting observation is that the values of conductancemeasured for CP1-4CSA obey monoexponential trend (see Fig. 4). Asindicated in the previous section, only CP3CSA and CP4CSA adoptwell-defined and quite rigid secondary structure, while CP1CSA andCP2CSA are less stiff. Thus, one could expect that the ability of theshorter and more flexible bridges to provide efficient pathway forelectron tunneling will be lower [59]. In order to explain such behavior,we need to consider the fact that the conductance of the junctions ismeasured while the molecules are slightly stretched. The tension mayresult in stiffening of the molecules entrapped between metalliccontacts and, this way, the molecule is tuned up to provide moreefficient conformation for electron tunneling. Such scenario seems tobe reasonable, since significantly higher conductance for rigid mole-cules containing cyclohexane rings comparing to their more flexiblen-alkyl analogues was reported for example by Martin and coworkers[59]. This would also support the assumption that the specific valuesof peptide dihedral angles may be responsible for efficient electroniccoupling of the neighboring units of the peptide bridge.

As an alternative, we have also considered the influence of the pri-mary structure on electron transfer enhancement. However, such possi-bility can be ruled out since recently, we have reported the conductancemeasurements for short oligopeptides of general formula Cys–AA–CSA(where CSA is cystamine, AA is Gly, Ala or Pro), which clearly showedthat electron transfer-mediating properties of these molecules are notaffected by the identity of the AA residue [60]. Moreover, the conduc-tance of Cys–AA–CSA was comparable to that observed for the simplealkanedithiol of similar length. Considering the results cited abovetogether with these reported in this paper, we can conclude that indi-vidual amino acids reveal similar electron transfer characteristics assimple alkyl bridges unless they form more rigid systems with well-defined conformation as reflected by relatively low value of β observedfor oligoprolines. Our results seem to confirm the theoretical predic-tions reported by Shin and co-workers, according to which polyprolineII bridges can provide very efficient pathway for long-range electrontransfer comparing to other secondary structures [61].

4. Conclusions

We have demonstrated that the conductance of thiolated oligo-proline derivatives trapped within the molecular junctions decays ex-ponentially with increasing length of the peptide backbone and the

26 J. Juhaniewicz, S. Sek / Bioelectrochemistry 87 (2012) 21–27

decay constant was 4.3 nm−1. Thus we conclude that electron trans-mission through short oligoprolines (up to four amino acid residues)is dominated by superexchange mechanism, which is in agreementwith earlier results reported in the literature [28–31,61]. Surprisingly,we have observed quite efficient tunneling although oligoprolines donot form intramolecular hydrogen bonds. The latter are usuallyconsidered as an important factor enhancing the electron transfer inpeptides. Based on these results, we have concluded that the confor-mation of the peptide backbone has primary effect on electron trans-fer efficiency and the role of the intramolecular hydrogen bonds as asupport for particular peptide structure may be more important thantheir ability to provide alternative electron transfer pathway.

Acknowledgements

This work was financially supported by the Ministry of Science andHigher Education (Grant no. N N204 138137). The project was alsooperated within the Foundation for Polish Science MPD Programmeco-financed by the EU European Regional Development Fund. Wethank Ewelina Zabost for help with CD measurements.

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