Sequence Specific Association of Tryptic Peptides with Multiwalled

Mar 14, 2012 - Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai, 400 005, India...
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Sequence Specific Association of Tryptic Peptides with Multiwalled Carbon Nanotubes: Effect of Localization of Hydrophobic Residues Megha S. Deshpande and Shyamalava Mazumdar* Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai, 400 005, India S Supporting Information *

ABSTRACT: A model-free approach has been used to study the association of peptides onto multiwalled carbon nanotubes (MWCNT) in aqueous solution at ambient pH to understand the molecular basis of interaction of the peptides with MWCNT. The peptides obtained by tryptic digestion of cytochrome P450cam from P. putida were allowed to interact with MWCNT, and several peptides were found to bind to the nanotube leading to formation of stable homogeneous dispersion of the bionano conjugates of MWCNT. The peptides bound to the MWCNT were separated from the unbound peptides and sequence analyses by tandem MS/MS technique identified the strongly bound peptides as well as the unbound and the weakly bound peptides. The peptide−MWCNT conjugate was further characterized by TEM as well as Raman, FTIR, vis-NIR absorption, and circular dichroism spectroscopy. A model based on the hydrophobicity of residues in the peptides suggested that the amphiphilic peptides with localized hydrophobic residues at the center or at one end of the sequence form stable dispersions of the peptide−MWCNT conjugates.

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inorganic surfaces.21 The peptides have also been shown to be useful in the fabrication of CNT-based nanodevices for use in electronic applications such as field-emission transistors, molecular-filtration membranes, optoelectronic devices, and sensors.22−33 Branched cationic and anionic peptides containing a short hydrophobic alkyl tail coupled to a more hydrophilic peptide sequence have earlier been used for noncovalent functionalization and dispersion of MWCNT in aqueous solution.30 The presence of phenylalanine in the series of amphiphilic helical peptides was shown to be important in the interaction between the peptides and SWCNT.34 Recently, PEGylated and N-fluorenyl-9-methoxycarbonyl-protected amino acid based surfactants have been successfully used for the dispersion of the carbon nanotubes.35,36 Most of the earlier reports, however, deals with single-walled carbon nanotubes (SWCNT), and very little work has so far been reported on association of specific peptides with MWCNT.30 The MWCNT are relatively easier to produce in large quantities compared to the SWCNT and they are more stable so that they can be potentially used for interesting bioapplications. However the MWCNT are intrinsically more inert and less soluble in aqueous solvents compared to the single-walled analogues.37 Moreover, MWCNT are generally larger in diameter and of higher density than single-walled nanotubes.37 Association of peptides may potentially help in dispersing the MWCNT in aqueous solutions. Therefore, screening of suitable peptides onto MWCNT to produce homogeneous dispersions of the nanotubes in aqueous solutions is necessary to understand the mechanism of recognition of the MWCNT by the peptides. Such studies would enable development of peptide-associated MWCNT that could have potential applications in biocompatible nanomaterials.

INTRODUCTION

Carbon nanotubes (CNT) have been widely studied due to their distinctive structural and physicochemical properties and diverse applications in material science and medicinal chemistry.1−9 However, the CNTs are highly hydrophobic and form insoluble aggregates in aqueous solutions rendering them difficult to assemble into applicable structures. Recently several advances in biological applications of carbon nanotubes have been reported, which includes the chemical modification of carbon nanotubes, highlighting specifically their covalent and noncovalent conjugates with a variety of biological and bioactive species. To fully explore their potential biological applications ranging from biosensors to pharmaceuticals, it is often required to increase the solubility of the CNTs in aqueous media through appropriate functionalization, which may be covalent or noncovalent in nature. To this end, noncovalent functionalization is particularly attractive to preserve the native structural and functional properties of CNTs. Aqueous dispersions of carbon nanotubes have been prepared with the use of surfactants, including detergents, polymers, and natural polysaccharides, such as gum arabic and amylose to separate carbon nanotube bundles.10−17 Separation of carbon nanotube bundles were shown to be improved by using additives like bile salts in addition to sodium dodecylsulfate (SDS) surfactants.18 However, traditional ionic surfactants, such as SDS and sodium dodecylbenzenesulfonate (SDBS), are toxic to mammalian cells.19,20 Therefore, to effectively and carefully introduce disaggregated pristine CNTs into biological systems, new biocompatible surfactants are needed. Thus, solubilization of the CNTs with suitable biocompatible agents still remains a big challenge.10 One possible approach to overcome such problems is the use of suitable peptides for the dispersion of CNTs. The peptides are generally less cytotoxic than detergents and are potentially easily degraded by cellular enzymes. Novel peptides have been designed for tailored binding to solids was reported using genetically engineered peptides that could recognize © 2012 American Chemical Society

Received: January 26, 2012 Revised: March 12, 2012 Published: March 14, 2012 1410

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We have developed a simple approach for selection of the specific peptides strongly bound to MWCNT from a mixture of peptides obtained by proteolytic digestion of a protein. The peptides derived from cytochrome P450cam from P. putida, which consists of a large number of aliphatic amino acids with hydrophobic side chain (A, I, L, V - 123) and hydrophobic amino acids (F, W, Y - 32), basic amino acids (R, H, K - 51), acidic amino acids (D, E - 54), amino acids with polar neutral side chain (N, C, Q, M, S, T - 99), and unique amino acids (G, P - 55) residues distributed in the protein structure, was taken as a model system for the present study. Proteolytic digestion of cytochrome P450cam with trypsin38 was used to produce a large number of peptides, which were assessed for affinity toward MWCNT. Detailed mass spectrometric analyses of the peptides helped in classifying the peptides based on their affinity to the MWCNT. Tandem MS/MS analyses led to identify the sequence characteristics of the peptides that are strongly associated with the nanotubes. The sequences of these peptides were distinctly different from the sequences of the peptides that are weakly bound or do not bind to the nanotubes. Various imaging and spectroscopic techniques such as TEM imaging, vis-NIR absorption spectroscopy, resonance Raman spectroscopy, FTIR, and circular dichroism (CD) spectroscopy, were exploited to characterize the peptide−MWCNT bioconjugates. Analyses of the residue-wise hydrophobicity of the peptides showed that localization of hydrophobic residues in the peptides that are strongly associated to the nanotubes. The studies demonstrated that the MWCNT bound to these peptides remain dispersed and stable in aqueous solution.

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then centrifuged for 50 min in a HERAEUS PICO21 centrifuge at 13000 rpm. The mixture was subjected to repeated ultrasonication and subsequent centrifugation that breaks the insoluble bundles of MWCNT and forms peptide−MWCNT conjugates dispersed in aqueous solution. The final supernatant containing the mixture of free peptides and peptide−MWCNT conjugate was collected and centrifuged for another 50 min to remove any remaining pristine MWCNT in solution. This procedure was repeated for two more times. The pellets containing unbound MWCNT were discarded. The supernatant containing the mixture of free peptides along with peptide−MWCNT conjugate was filtered through a centricon tube (10 KD cutoff membrane filter) that separated the peptide−MWCNT conjugate from free peptides. This procedure was repeated for one more time. The solution of unbound peptide was collected and concentrated for sequence identification. The peptides bound to the nanotubes dissolves in acetonitrile and the solution filtered through the syringe filter remove the MWCNT from the conjugate. The filtrate, thus, consists of the bound peptides from the peptide− MWCNT conjugate, which was collected and concentrated for sequence identification of the bound peptides. Liquid Chromatography: MS/MS Analyses. Liquid chromatography coupled to mass spectrometric (ESI-LCMS) analyses of the peptides were performed on a Thermo Finnigan LCQ Deca Electrospray quadrupole ion trap mass spectrometer (ThermoElectron Co., Hemel Hempstead, Herts, U.K.) with a Thermo Surveyor high performance liquid chromatography (HPLC) pump and microelectrospray source and operated with Thermo Xcalibur software, version 1.4. Peptides were resolved with an 11 cm, 100 μm ID, C18 (Column Engineering) microcapillary column with a 5−15 μm tip opening. The flow rate from the HPLC pump was adjusted to achieve 1 mL/min. The gradient was raised to 2% acetonitrile after 10 min, which increased to 60% at 45 min, 95% at 45.2 min, then to 95% at 55 min before decreasing to 2% at 55.2 min. The total analysis time for this method was 70 min. All MS analyses were performed in positive ion mode. The tandem mass (MS/MS) spectra of the peptide fragments were analyzed by Turbo-Sequest software (ThermoElectron Co.). Visible-Near-Infrared (Vis-NIR) Spectroscopy. Absorption spectra of peptide−MWCNT conjugate as well as of the sonicated suspension of pristine MWCNT in water were acquired using a Perkin-Elmer (Lamda 750) UV−vis-NIR spectrophotometer at room temperature. Transmission Electron Microscopy (TEM). TEM images were acquired using a FEI Tecnai 20 Transmission Electron Microscope at an accelerating voltage of 200 kV. One drop of an aqueous peptide− MWCNT conjugate solution was placed on a 200 mesh carbon support grid. The TEM grid was kept overnight for completely drying in the air before TEM measurements were conducted. The control sample was the sonicated suspension of pristine MWCNT in water that was drop casted on the carbon grid as was done for the peptide− MWCNT conjugate. Raman Spectroscopy. Raman spectra were taken using a Horiba Yvon Raman Spectrometer T64000, which has an inverted optical microscope adapted to a triple-grating equipped with (1024 × 256) liquid N2 cooled CCD. A spectra-Physics model with Argon Ion laser provided the excitation at 514.5 nm. The laser power at the sample was 200 mW at the laser head and was focused using a 10× objective lens. Wavenumber calibration was carried out using the 521 cm−1 line of the silicon wafer. Raman analyses on peptide−MWCNT conjugate supernatants (∼10 μL) were acquired from three different sample preparations to ensure reproducibility. Spectra were recorded by scanning the 1200−1800 cm−1 region with a total acquisition time of 30 min. Spectra were fitted with Lorentzian functions by searching for the minimum number of frequencies that fit the different bands equally well without fixing the positions and widths of the individual peaks. Fourier Transform Infrared Spectroscopic Studies. Infrared spectra were recorded on a JASCO FTIR-4100 Spectrometer at room temperature for mid-IR range (400−4000 cm−1). The thin films of the samples were prepared on a CaF2 plate under nitrogen atmosphere immediately before experiment.

EXPERIMENTAL SECTION

Materials and Reagents. Dithiothreitol (DTT) and Trypsin Gold were purchased from Promega, India, HPLC grade water, acetonitrile, methanol, trifluoroacetic acid, ammonium bicarbonate, and urea were obtained from Merck Chemicals, India, DEAE Sepharose, QSepharose, and Sephadex G-25 and PD10 (Sephadex G25) columns were from GE Healthcare Biosciences AB (Uppsala, Sweden), and Centricon concentrators (10kD cutoff) were obtained from Millipore, India. All general reagents and (1R)-camphor and MWCNT were from Sigma Aldrich. Enzymatic Digestion of Cytochrome P450cam. The wild-type cytochrome P450cam was expressed and purified using reported procedure.39−41 The glycerol stock solution of cytochrome P450cam was desalted using PD10 desalting column (Sephadex G-25) and the solution then concentrated using centricon tube (Millipore). A concentrated solution of cytochrome P450cam (50 μL) was used for the enzymatic digestion. To allow full access by proteolytic enzyme, denaturation of protein was carried out in the presence of reducing agent DTT (200 mM; freshly prepared in ammonium bicarbonate buffer 100 mM, pH 8.5) to prevent cross-linking of cystein residues and 4 M urea (50 μL) and 30% methanol was added to improve the sequence coverage. The reaction was carried out at 60 °C for 1 h to allow the reduction of disulfide bridges. Vial was then kept at 37 °C for cooling. Further 500 μL of ammonium bicarbonate (100 mM, pH 8.5) was added to the solution to dilute the urea concentration. The vial was then vortexed for few minutes. Finally, digestion of protein is carried out in the presence of trypsin with the ratio of trypsin to protein 1:25 (1 mg of trypsin for every 25 mg of protein to be digested). The optimal pH for trypsin is in the range from 7.0 to 9.0. Digestion was carried out at 37 °C for different time intervals and the best data was obtained after 16 h digestion. The sample was cooled and dried. A 50 μL portion of the digested protein solution with the addition of 100 μL of 50% acetonitrile in water was further used for mass spectrometric analysis. Preparation of Peptide Multiwalled Carbon Nanotube Conjugate (Peptide−MWCNT Conjugate). Peptide−MWCNT conjugate preparation was carried out by slight modification of the literature procedures.1,34,42,43 The ∼2 mg MWCNT was added to 500 μL of aqueous solution of the tryptic peptide mixture in an eppendorf tube. The suspension was subjected to ultrasonication for 1 min using a Hielscher UP400S Ultrasonic Processor equipped with a 3 mm diameter micro tip. The solution was allowed to stand overnight and 1411

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Circular Dichroism Spectroscopic Studies. Circular dichroism (CD) spectra in the secondary-structure region (190−260 nm) were recorded in a quartz cuvette of 1 mm path length using a JASCO J810 Spectropolarimeter (JASCO Corporation, Tokyo, Japan). CD was performed on the intact protein and the mixture of peptides from cytochrome P450cam in aqueous solution, peptide−MWCNT conjugate and the unbound peptide solution. The spectra were measured between 190 to 260 nm with the average of three successive scans. The secondary structures of the protein and peptide were estimated by the in-built software provided by JASCO using YANG’s method. Hydrophobicity Calculation for the Peptide Sequences. The hydrophobicity parameters for the bound, unbound and weakly bound peptides were estimated using the application server in the Expasy site (http://www.expasy.ch/tools/protscale.html). Hydrophobicity of amino acids was calculated based on the Eisenberg et al. method.44 The projections of the amino acids in the peptide sequence assuming a β-sheet conformation of the MWCNT-bound peptides were calculated using the pepwheel (http://150.185.138.86/cgi-bin/emboss/ pepwheel) server program from the European Molecular Biology Open Software Suite (EMBOSS). The parameters used in the pepwheel analyses for β-sheet conformation were nine steps for four turns corresponding to two residues per turn.

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Figure 1. (a) MS plot for the peptide with sequence PLVWDPATTKAV obtained after tryptic digestion of cytochrome P450cam showing peak with mass 1298.12 (R.T. = 34.15); (b) MS/MS plot for the same peptide with +1 charge state (only b- and y-type ions indicated for the sake of simplicity).

RESULTS AND DISCUSSION

Enzymatic Digestion of cytochrome P450cam. Trypsin is commonly used to produce specific peptides on digestion of proteins at ambient pH.45 In the present study, cytochrome P450cam was used as the source of the peptides that were studied for the association with MWCNT. The peptides produced by trypsin digestion of cytochrome P450cam were separated by high pressure liquid chromatography (HPLC), and the peptide sequences were analyzed by ESI-MS/MS using the Turbo-Sequest software. In the peptide identification process, search were performed for the tryptic peptides, that is, those peptides that were formed by cleavage after a basic amino acid residue (arginine or lysine) except when the basic residue was followed by a proline. This covers ∼50% of the protein sequence.38 Search was also performed for the identification of nontryptic/semitryptic peptides, which are truncated from one end of the tryptic peptide, thus preserving one trypsin cleavage site.38 The peptides produced by digestion of cytochrome P450cam with trypsin were found to cover ∼75% of the sequence of the protein. The results of the ESIMS/MS study identifying the peptide sequences with their average mass and sequence position are summarized in the Table S1 in the Supporting Information. The typical MS spectrum for a peptide with sequence PLVWDPATTKAV obtained by digestion of the enzyme is shown in Figure 1a. The tandem MS/MS spectrum of this peptide is shown in Figure 1b. Analyses of the MS/MS data by Turbo-Sequest using a nonredundant (NR) protein database assigned several fragment ions (Table S2 of the Supporting Information) of the type bn(m +), yn(m+), an(m+), and xn(m+) (where n = 1−12 and m = 1), which allowed us to identify the sequence of the individual peptide. Screening of Peptides Based on Affinity to MWCNT. The preparation of the peptide−MWCNT conjugate was carried out as described in the Experimental Section. The aqueous solution containing the peptide−MWCNT conjugate was highly stable for several months and was well dispersed, while that of pristine MWCNT quickly forms black precipitate of the nanotubes (Figure 2). The separated solution of the peptides bound with MWCNT and that of the unbound peptides were separately analyzed by ESI-LCMS for the peptide

Figure 2. Photograph of tubes showing (a) peptide−MWCNT conjugate aqueous solution and (b) pristine MWCNT in water. Differences among the peptide−MWCNT conjugate dispersions were visually observed by a difference in color intensity.

sequence identifications. The list of the peptides bound with MWCNT and that of the unbound peptides are given in the Tables 1 and 2, respectively. There were 12 unique peptide sequences identified in the acetonitrile extract of the peptide− MWCNT conjugate solution that were absent in the solution of unbound peptides, indicating that these peptides are strongly bound to the MWCNT (Table 1). The binding affinities of these strongly bound peptides for the MWCNT are possibly very high so that no residual peptide of this category could be detected by LCMS in the filtrate obtained by Centricon concentrator (10kD cutoff). There were 11 unique peptide sequences detected in the solution of the unbound peptides that were not found in the peptide−MWCNT conjugates, suggesting that these peptides do not have any affinity for the nanotube (Table 2). We also detected seven peptide sequences in the acetonitrile extract of peptide−MWCNT that were also present in the solution of unbound peptides, indicating that these peptides are weakly bound to the nanotube (Table 3). 1412

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Table 1. Assignment of the MWCNT Bound Peptide (BP) Sequencesa No.

peak mass

peptides sequence

peptide position

BP1 BP2 BP3 BP4 BP5 BP6 BP7 BP8 BP9 BP10 BP11 BP12

1373.5 1954.3 1988.3 1458.5 1926.1 2511.8 1779.0 1384.7 1780.0 1950.3 704.8 1298.5

NPSNLSAGVQEAW ALANQVVGMPVVDKLENR LENRIQELACSLIESLR RPQGQCNFTEDY QKPGTDAISIVANGQVNGR PGTDAISIVANGQVNGRPITSDEAK ILTSDYEFHGVQLKK KKGDQILLPQML SRQKVSHTTFGHGSHL GQHLARREIIVTLKEW EWLTR PLVWDPATTKAV

31−43 114−131 128−144 144−155 214−232 216−240 301−315 314−325 342−357 360−375 374−378 404−415

Table 3. Assignment of the MWCNT Weakly Bound Peptide (WBP) Sequencesa No.

peak mass

peptide sequence

peptide position

WBP1 WBP2 WBP3 WBP4 WBP5 WBP6 WBP7

1017.1 678.8 1021.2 1384.7 1786.1 1996.3 823.9

PITSDEAKR RRFSL RFSLVADGR KKGDQILLPQML GDQILLPQMLSGLDER SRQKVSHTTFGHGSHLCL GHGSHLCL

233−241 291−295 292−300 314−325 316−331 342−359 352−359

a

Polar residues are shown in bold and aromatic residues are shown in italics.

a

Polar residues are shown in bold and aromatic residues are shown in italics.

The typical MS spectrum for the MWCNT bound peptide (sequence No. BP1, Table 1) with sequence NPSNLSAGVQEAW is shown in Figure 3a. The tandem MS/MS spectrum of this peptide with a +2 charge state is shown in Figure 3b. Analyses of the MS/MS data as discussed above led to identifying the sequence of the individual peptides (Table S3 of the Supporting Information). Figure 3b shows the CID of the peptide NPSNLSAGVQEAW in the tandem MS/MS study. Analogously, the typical MS spectrum for the MWCNT unbound peptide (sequence No. UBP1, Table 3) with sequence DMYNPSNLSAGVQEAW is shown in Figure 4a. The tandem MS/MS spectrum of this peptide with a +3 charge state is shown in Figure 4b (Table S4 of the Supporting Information). The MS/MS spectra for the bound and unbound peptides investigated by ESI-LCMS analysis are given in the Supporting Information (Figures S1 and S2). It is important to note that the ESI-LCMS studies could not detect any peptide formed by autolysis of trypsin in the peptide mixture in the present case. The mass spectrometric identification of bound, unbound, and weakly bound peptides correspond to the proteolytic products of cytochrome P450cam only. The concentration of trypsin was very small compared to that of cytochrome P450cam, and trypsin gold used in the present study is also known to be relatively more resistant to autolysis compared to the native trypsin (Porcine). Absence of any mass spectroscopic signature of trypsin autolysis in the present studies possibly indicates that

Figure 3. (a) MS plot for the MWCNT bound peptide (BP1) with sequence NPSNLSAGVQEAW showing a peak with mass 687.56 (R.T. = 53.58); (b) MS/MS plot for the same peptide with +2 charge state.

the autolysis products, if any, might be beyond the detection limit of the mass spectrometer. Vis-NIR Spectroscopy. The repeated ultrasonication and subsequent centrifucation of the mixture of tryptic peptides containing the MWCNT leads to formation of stable aqueous dispersion of peptide-MWCNT conjugates. Vis-NIR spectroscopy was used to identify the dispersion of the carbon nanotubes.1,28,34 The vis-NIR absorption spectra of the aqueous dispersion of peptide−MWCNT conjugate is shown in Figure

Table 2. Assignment of the MWCNT Unbound Peptide (UBP) Sequencesa

a

No.

peak mass

peptide sequence

peptide position

UBP1 UBP2 UBP3 UBP4 UBP5 UBP6 UBP7 UBP8 UBP9 UBP10 UBP11

1782.9 1600.8 1394.5 2266.6 1497.8 2117.5 1410.6 2315.6 454.5 1501.8 3644.2

DMYNPSNLSAGVQEAW LTDQMTRPDGSMTF SPEHRQELIER QELIERPERIPAACEELLR PERIPAACEELLR IPAACEELLRRFSLVADGR SLVADGRILTSDY VSHTTFGHGSHLCLGQHLARR GQHL EIIVTLKEWLTR IPDFSIAPGAQIQHKSGIVSGVQALPLVWDPATTK

28−43 181−194 268−278 273−291 279−291 282−300 294−306 346−367 360−363 367−378 379−413

Polar residues are shown in bold and aromatic residues are shown in italics. 1413

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this region, and the absorbance of the MWCNT dispersed in triton-X-100 was found to increase with an increase in concentration of the surfactant analogous to that reported in the case of SWCNT.46 These results support that the peptides can more efficiently disperse the MWCNT than the surfactant. Moreover, the peptide−MWCNT conjugate dispersion was stable for several months, while the MWCNT dispersed in triton-X-100 precipitates within a few days, forming aggregates of MWCNT. This effect can be explained in terms of both the sequence and the structure of the peptides (see later). Different peptides coat MWCNT through noncovalent interactions (π−π stacking, van der Waals, and hydrophobic interactions), thereby enhancing MWCNT dispersion. Transmission Electron Microscopy. To further confirm that the peptides indeed coat on the MWCNT, we studied the dispersions of pristine MWCNT in the surfactant (triton-X100) and that of peptide−MWCNT conjugate by transmission electron microscopy (TEM). A clear difference was observed between the solutions of MWCNT in the absence and in the presence of peptides as shown in Figure 5. The overall thickness of the pristine MWCNT solubilized in dilute tritonX-100 was about 7−15 nm (Figure 5a). Unlike in the case of SDS solubilized SWCNT,47 the MWCNT solubilized in dilute triton-X-100 did not show any clear signature of formation of coating of organic molecules on the nanotubes. On the other hand, the peptide−MWCNT conjugate dispersion on a TEM grid revealed an extensive network of nanotubes coated with peptides forming a web-like film (Figure 5b). A coating of the peptides was observed on the MWCNT in the presence of peptides. Similar results were earlier reported in case of MWCNT coated with branched anionic and cationic peptides,30 and SWCNT coated with amphiphilic peptide helices.42 The thickness of the peptides coating on the MWCNT in the present case were found to vary from 0.7 to 15 nm along the length of the MWCNT. This irregularity in thickness suggests that the different bound peptides form multiple layers with varying orientation on the MWCNT surface, as reported earlier.30

Figure 4. (a) MS plot for the MWCNT unbound peptide (UBP1) with sequence DMYNPSNLSAGVQEAW showing a peak with mass 594.69 (R.T. = 48.63); (b) MS/MS plot for the same peptide with +3 charge state (only b- and y-type ions indicated for the sake of simplicity).

S3 (see the Supporting Information) along with that of the pristine MWCNT suspended in water and the aqueous dispersion of unmodified pristine MWCNT in triton-X-100. The presence of well-defined absorbance peaks at ∼950 and ∼1150 nm region indicate the presence of isolated MWCNT for the peptide−MWCNT conjugate dispersion. These peaks correspond to optical interband transitions within one-dimensional density of states (DOS) in the nanotubes as reported earlier for SWCNT.30 The observation of a broad absorption band in the MWCNT instead of sharp bands characterizing van Hove singularities reported in the case of SWCNT may indicate the presence of a large number of closely lying states in the multilayer structure of the MWCNT. The aqueous dispersion of the bundled MWCNT did not show any absorption band in

Figure 5. TEM micrographs of (a) pristine MWCNT in the absence of peptide at low magnification (bar = 100 nm) dispersed in dilute triton-X-100 aqueous solution; (b) peptide−MWCNT conjugate aqueous solution at low magnification (bar = 100 nm). 1414

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Raman Spectroscopy. Raman spectroscopy is a powerful nondestructive technique for the study of the structure, especially for the identification of doping and disorder in carbon nanostructures.48−50 We have investigated the effects of the association of the peptides on the MWCNT (Figure 6) by

Figure 6. Raman spectra showing the “D”- and “G”-band features for pristine MWCNT (dash line) and peptide−MWCNT conjugate aqueous solution (solid line).

Raman spectroscopy of the modified and unmodified nanotubes. The Raman spectrum of the CNT is characterized by two distinct bands, namely, the D- and G-bands. The intensity of the D-band is used to probe defects or disorder in the surface of the carbon nanotubes. The G-band corresponds to doubly degenerate phonon mode of the sp2 carbon network and is characteristic of the sp2 framework. Changes in the intensity as well as position of the G-band are used to probe noncovalent interactions that may affect the electron density on the graphene surface of the nanotube. Figure 6 shows the characteristic D- and G-bands for pristine MWCNT and the peptide−MWCNT conjugate. The D- and G-bands in pristine MWCNT appear, respectively, at 1348.6 and 1591 cm−1. The peptide−MWCNT conjugate shows Raman bands at 1348.6 and 1627 cm−1 corresponding to the D- and G-band, respectively. The results in Figure 6 show that the position of the D-band does not change on association of the peptide to the MWCNT supporting the noncovalent functionalization of MWCNT by the peptides. A clear upshift of ∼36 cm−1 in the spectral position of the G-band of the MWCNT associated with the peptides indicates adsorption of the peptides along the outer surface of the MWCNT. The upshift in the G-band may arise due to various noncovalent interactions including van der Waals, hydrophobic, π-stacking, as well as weak charge transfer interactions between the π-electron of the CNT as the donor and the adsorbent as the acceptor.48 Previous studies also reported that the tangential vibrational modes (G-band) for carbon nanotubes are sensitive for noncovalent doping.49,50 These results thus support that the strongly bound peptides possibly have specific noncovalent interactions with the MWCNT that cause the upshift of the G-band in the present case. FTIR Spectroscopy. The nature of interaction of MWCNT with peptides from cytochrome P450cam was further investigated by Fourier transform infrared spectroscopy (FTIR). The infrared spectra of the mixture of tryptic peptides and that of the purified the peptide−MWCNT conjugate (separated from the unbound peptides) are recorded at room temperature in the range 400−4000 cm−1 (Figure 7a,b). The amide I and II bands are the two major bands in the infrared

Figure 7. FTIR spectra of (a) mixture of tryptic peptides from cytochrome P450cam; (b) peptide−MWCNT conjugate (Insets: enlarge spectra 1000−1800 cm−1).

spectrum of the peptides that are sensitive to the conformation of the peptide backbone.51,52 The amide I band in the range 1600−1700 cm−1 was earlier shown to be associated with the CO stretching vibration and is directly related to the backbone conformation.51,52 The insets of Figure 7 show the FTIR spectrum in the range 1000−1800 cm−1 and the positions of the major infrared bands for the mixture of tryptic peptides and those of the peptide−MWCNT conjugate are given in Table 4. The amide I region of the FTIR spectrum of the peptides is broad, indicating the presence of heterogeneous conformations of the amides. The mixture of all tryptic peptides from cytochrome P450cam shows distinct indications of the bands at 1621 and 1649 cm−1 (Figure 7a, Table 4), which are characteristic of the β-sheet and random coil conformations of the peptides.51,52 The FTIR spectrum of the isolated peptide− MWCNT conjugates (i.e., free from unbound peptides) in the amide I region is upshifted compared to that of the mixture of the tryptic peptides and indicated presence of bands at ∼1627, ∼1636, ∼1647, and ∼1654 cm−1 (Figure 7b, Table 4). The comparison of the spectrum of the nanobioconjugate with that of the mixture of the tryptic peptides indicates that the amide I bands of the peptides (Figure 7a, Table 4) are upshifted in the peptide−MWCNT conjugate (Figure 7b, Table 4) possibly because of the formation of rigid conformations containing the β-sheet structures on association with the nanotubes.51 The FTIR spectrum of the peptide−MWCNT in the 1600−1700 cm−1 region also indicates the presence of a band at ∼1647 cm−1, which may arise from the random coiled conformations 1415

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Table 4. FTIR Spectroscopic Properties of the Mixture of Tryptic Peptides and Peptide−MWCNT Conjugate FTIR spectroscopic results band assignment

mixture of tryptic peptides (cm−1)

peptide−MWCNT conjugate (cm−1)

amide I (80% CO stretch) amide II (60% N−H bend and 40% C−N stretch) aromatic nature (CC) amide C−N stretch amide III (very weak 40% C−N stretch and 30% N−H bend) weaker band results from interaction between N−H bending and C−N stretching aromatic in plane C−H bending/Symm. C−O−C assigned conformation

1621 and 1649 1528 1446 1409 near 1300 1223 1045 β-conformation or random conformation

1627, 1636, 1647, and 1654 1543 1457, 1647 1414 near 1300 1226 1041 β-conformation

designed peptide)34 or unfolded, unstructured states (peptide phage selection),32 depending on the nature of the peptide. Earlier reports also suggested that the conformation of the carbon-nanotube bound state of the polypeptide was similar to that in the solution state,34 which does not seem to be applicable in the present case. Recently, the importance of the tryptophan residue in the formation of a SWCNT-binding peptide with the motif, XTHXXPWTX and SXWWXXW (X is any amino acid) were reported.33,55 The structure of aromatic rings of the amino acid was proposed to stack on the graphene surface that may decrease the effective hydrophobic surface of the peptides exposed to the polar solvent. The van der Waals interaction between the peptide and the nanotube would also be enhanced in the aromatic residue containing peptides. Earlier computational studies on binding free energies between peptides and SWCNT provided a framework for understanding of the binding strength and indicated that van der Waals interaction possibly plays a key role in the binding of peptides to the CNT.56 The present results indicate that the interaction of the nanotube may also affect the conformation of the peptide bound to it so that the most stable structure of the nanobio conjugate is formed. Analyses of Hydrophobicity of the Residues. Recent studies on interaction of peptides with SWCNT32,33,55,57−59 suggested that the peptides with significant “surfactant-like” behavior may preferentially associate with the hydrophobic surface of the nanotube and help in dispersing the nanotube in aqueous solutions. Histidine and tryptophan were proposed to be important for binding to SWCNT.55 MWCNT consists of several layers of the nanotubes with larger outer diameter and thus may require stronger stabilization interaction than that for SWCNT to disperse in aqueous medium. The sequences of the peptides that are strongly bound to the MWCNT in the present case showed localization of the hydrophobic residues or presence of aromatic residues in the peptide sequence. The plot of residue-wise hydrophobicity44 of the peptides bound to the MWCNT (Figure 8a, also see Figure S5 in Supporting Information) indicates that the hydrophobic residues are localized either at the center or at one end of the peptide suggesting specific “amphiphilic” property (Figure S5, Supporting Information). On the other hand, analogous plot for the unbound peptides (Figure 8b, also see Figure S6 in Supporting Information) did not show any localization of hydrophobicity. The analyses of the residue-wise hydrophobicity of the peptides thus seem to support that the peptides with localized hydrophobicity would tend to self-assemble forming hydrophobic patch and associate on the nonpolar and hydrophobic surface of MWCNT to minimize the interfacial energy of the nanotube−water interface with the polar residues of the peptide

of the peptides as well as from the aromatic CC vibrations of the MWCNT.51,53 The band at 1528 cm−1 (amide II) of the mixture of the tryptic peptides (Figure 7a) represents side chain vibrations, which was upshifted to 1543 cm−1 in the case of the peptides associated to the MWCNT (Figure 7b), supporting predominant β-sheet conformation in the MWCNT-bound peptides. In addition to these, the peptide mixture also shows bands at 1045, 1223, ∼1300, 1409, and 1446 cm−1 assigned to various modes of vibrations in the peptides (Table 4),54 which were almost unaffected on association of the peptides to the nanotubes. The FTIR results shown in Figure 7 thus show that the peptides are indeed bound to the nanotubes and that the MWCNT-bound peptides have predominantly β-sheet conformation. Circular Dichroism Spectroscopic Studies. To assess the conformational properties of the peptides and the effects of the association of the peptides to the MWCNT, we carried out farUV circular dichroism (CD) studies of the tryptic peptide mixture from cytochrome P450cam, the peptide−MWCNT conjugate and the peptide solutions that did not bind to the MWCNT (unbound peptides; Figure S4 in the Supporting Information). The secondary structures of the peptides estimated from the CD results are tabulated in Table 5. The Table 5. CD Spectroscopic Properties of the Mixture of Tryptic Peptides and Peptide−MWCNT Conjugate CD spectroscopic results sample

% α-helix

mixture of tryptic peptides peptide−MWCNT conjugate

25.8 (minor) 28 (minor)

% βconformation

% random conformation

% β-turn

48.9 (major)

25.3 (minor)

72 (major)

results show that the mixture of tryptic peptides from cytochrome P450cam exhibits a predominantly random coil conformation (∼50%) along with α-helix conformation (∼26%). The peptide−MWCNT conjugate showed distinctly different CD results and the MWCNT bound peptides were found to adopt predominant β-sheet conformation (∼72%), while α-helix content (∼28%) remained almost the same as that in the total peptide mixture obtained from cytochrome P450cam. The CD results thus support that the conjugation of the peptides with MWCNT possibly leads to the formation of the β-sheet conformation of the bound peptides, while the unbound peptides had predominantly random coiled conformation. Earlier reports have shown that certain carbon nanotube specific peptides may adopt an α-helix (rationally 1416

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acids in the peptide sequence were calculated using the pepwheel (http://150.185.138.86/cgi-bin/emboss/pepwheel) server program (Figure S8, Supporting Information). The results of the pepwheel analyses (considering β-sheet conformation, corresponding to nine steps and four turns) show that most of the bound peptides indeed consist of a hydrophobic patch, which agrees with earlier reports.33,60 Over and above, these results suggest that the hydrophobic interactions and π-stacking interactions between the MWCNT and the peptide play an important role for the association of the peptides onto the nanotubes, while the polar residues in the peptide helps in solubilization of the nanobioconjugate. The present results thus suggest that the peptides predominantly in the β-sheet structure tend to cluster together so that the hydrophobic residues form a patch that can strongly associate onto the surface of the MWCNT, while the polar residues in the peptides help the bionanoconjugate to disperse in aqueous solution. A schematic structure based on semiempirical modeling (Hyperchem 6) of the assembly of the peptides on the SWCNT is given in Figure 9, which shows

Figure 8. Hydrophobicity (calculated by method of Eisenberg et al.) plot for (a) the peptide sequence QKPGTDAISIVANGQVNGR (BP5) that strongly binds to MWCNT, (b) the peptide sequence IPAACEELLRRFSLVADGR (UBP6) that does not bind to MWCNT, and (c) the peptide sequence GDQILLPQMLSGLDER (WBP5) that binds weakly to MWCNT.

directed to the solvent making peptide−MWCNT conjugate dispersed in aqueous solution.33,55,57−59 Figure 8a shows the calculated hydrophobicity plot of a typical bound peptide (eg., sequence QKPGTDAISIVANGQVNGR (BP5) that strongly binds to MWCNT) and Figure 8b shows that of a typical unbound peptide (eg., sequence IPAACEELLRRFSLVADGR (UBP6) that does not bind to MWCNT). The tryptic peptides obtained from cytochrome P450cam in the present case also showed that there are seven weakly bound peptides that are present both in the solution of unbound as well as of bound peptides and they were found to have very similar type of distribution of hydrophobic residues (Figure S7, Supporting Information) as that found in the bound peptides (Figure 8c, for example, the peptide sequence GDQILLPQMLSGLDER (WBP5) that binds weakly to MWCNT). Thus, the present results indicate that analyses of residue-wise hydrophobicity of the peptides may provide a qualitative assessment of the propensity of binding of a peptide to the MWCNT. Earlier studies by Wang et al.32 used phage display to screen peptides associated with SWCNT and showed that the CNT binding sequence of the peptides consists of amphiphilic character analogous to that observed in the present case. It is, however, important to note that the bound peptides acquire definite conformations when they are associated onto the MWCNT. Recent studies by Zheng et al.,33 showed by helical wheel diagram, that the SWCNT binding peptides had hydrophobic and aromatic side groups directed toward one side of the peptide to enable effective hydrophobic binding of the peptide to the nanotube. Computational studies and restricted screening of helical peptides by combinatorial chemistry methods were shown to stabilize the SWCNT−peptide bioconjugates by geometrically defined molecular surfaces of the peptides.60 The nature of the surface of the peptide would depend on the specific conformation of the peptide in such case. Considering a β-sheet conformation of the MWCNTbound peptides, as indicated from the FTIR and CD studies in the present case, the corresponding projections of the amino

Figure 9. Schematic structure based on semiempirical modeling of the assembly of the peptides on the SWCNT.

wrapping of the peptide around the carbon nanotubes so that the β-sheets of the peptides lie parallel or antiparallel with respect to each other and form oligomers that are possibly stabilized by peptide−nanotube and peptide−peptide interactions.

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CONCLUSIONS The screening of the peptides bound onto MWCNT from a mixture of tryptic peptides of cytochrome P450cam provided a simple route for obtaining 12 peptide sequences that were strongly associated to the nanotubes. Cytochrome P450cam is chosen in the present study as it consists of a significant fraction of hydrophobic residues distributed all over the protein. However, one could apply similar approach to screen peptides from any large protein for binding to carbon nanotubes. The present studies on MWCNT further supported the earlier 1417

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results on SWCNT and demonstrated that the surfactant like property of the peptide for association of the hydrophobic surface of the nanotube is essential to form stable dispersions of the MWCNT in solution. The Raman spectroscopic results establish the noncovalent interaction between MWCNT and the peptides. The experimental results suggest that MWCNT− peptide conjugate had predominant β-sheet structure and have excellent hydrophobic−hydrophilic balance that is suitable for the noncovalent functionalization, good dispersion stability of the MWCNT−peptide conjugate. The design criteria employed herein can also be applied to the coating of other nanomaterials and more functionalities can be engineered into the peptide by inserting other peptide domains, which can further used in synthesis of nanoconjugates and heterostructures for a variety of applications.

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ASSOCIATED CONTENT

S Supporting Information *

Table for the observed peptide sequences after an enzymatic digestion of cytochrome P450cam by trypsin (Table S1); the fragment ions observed for the peptide with sequence PLVWDPATTKAV obtained after tryptic digestion of cytochrome P450cam (Table S2); the fragment ions observed for the MWCNT bound peptide (BP1) with sequence NPSNLSAGVQEAW (Table S3); the fragment ions observed for the MWCNT unbound peptide (UBP1) with sequence DMYNPSNLSAGVQEAW (Table S4); MS/MS spectra for MWCNT bound peptides from cytochrome P450cam (Figure S1); MS/MS spectra for MWCNT unbound peptides from cytochrome P450cam (Figure S2); the absorption spectra of pristine MWCNT, peptide−MWCNT conjugate, and triton-X100-treated MWCNT (Figure S3); CD spectra of mixture of peptides from cytochrome P450cam, bound peptide− MWCNT conjugate solution, unbound peptides, and cytochrome P450cam enzyme (Figure S4); hydrophobicity plot for the MWCNT bound, unbound, and weakly bound peptides (Figures S5−7). β-Sheet conformation of the strongly bound peptides (BP1−12) to MWCNT by using pepwheel drawing (Figure S8). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 009122 22782363. Fax: 009122 2280 4610. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by the Tata Institute of Fundamental Research, Mumbai, India, and the Department of Biotechnology (DBT), India. The authors wish to thank Bharat Kansara for technical help in the ESI-LCMS studies, Dr. Shankar Ghosh and Smita Gohil for help in the Raman Experiments, and Dr. Shashank Purandare, Rudheer Bapat, and Bhagyashree Chalke for the TEM Measurements. Authors wish to thank Prof. K. L. Narasimhan for the IR experiments.

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