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Modified Filamentous Bacteriophage as a Scaffold for Carbon Nanofiber Katarzyna Szot-Karpi#ska, Piotr Golec, Adam Lesniewski, Barbara Palys, Frank Marken, Joanna Niedzió#ka-Jönsson, Grzegorz Wegrzyn, and Marcin #o# Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00555 • Publication Date (Web): 17 Oct 2016 Downloaded from http://pubs.acs.org on October 18, 2016

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Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

Graphical abstract. Schematic illustration of carbon nanofibers (CNF) binding by the M13 phage with point mutation in pVII protein (pVII-mutant-M13).

196x155mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Table 1. Comparison of changed region of pVII (nucleotide and amino acid sequences) of the unmodified phage (pVII-M13) and modified phage with point mutation (pVII-mutant-M13). 128x39mm (300 x 300 DPI)


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Fig. 1. Efficiency of (a) CNP and (b) CNF binding by the phage (pVII-mutant-M13) with point mutation on the pVII protein and by the unmodified phage (pVII-M13). The presented results are average values from four experiments with RSD represented by error bars. 59x20mm (300 x 300 DPI)



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Table 2. The average values of the calculated binding efficiency (O/I - ratio of the output phage number (phages eluted/O) to the input phage number (phages incubated/I with target material)) obtained for studied carbon nanomaterials and the pVII-mutant-M13 phage vs. pVII-M13. The presented results are average values from four experiments with RSD represented by error bars. 54x30mm (300 x 300 DPI)


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Fig. 2 AFM images of CNF (a, b) specifically bound to the modified protein of the pVII-mutant-M13 and (c, d) unbound with the unmodified protein of the pVII-M13 (c,d). AFM (a) amplitude error and (b-d) topographic images. Due to large difference between scales of phages and CNF particles, the above images contain only fragments of CNF particles.



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Fig. 3 TEM images of CNF (a, b) specifically bound with the pVII-mutant-M13 and (c, d) with the unmodified pVII-M13 bacteriophages visibly unbound. 196x152mm (300 x 300 DPI)


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Fig. 4 Absorbance spectra for pVII-mutant-M13 solution (black line) and CNF-pVII-mutant-M13-CNF (blue line). The shift of the peak at about 230 nm marked with red area. 58x40mm (300 x 300 DPI)



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Fig. 5 TEM images of carbon nanomaterials (a) graphite (Gr), (b) reduced graphene oxide (rGO), (c) singlewalled carbon nanotubes (SWCNTs), (d) multi-walled carbon nanotubes (MWCNTs) and their interactions with the pVII-mutant-M13 phages. 196x144mm (300 x 300 DPI)


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Fig. 6 SEM images of CNF (a) with the pVII-mutant-M13 (b) and the pVII-M13 (c) materials deposited on the ITO electrode.



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Fig. 7 Infrared spectra of CNF with the pVII-M13 (CNF-pVII-M13), pVII-mutant-M13 (CNF-pVII-mutant-M13) and the pVII-mutant-M13 without CNF material deposited on the on silica surface. Inset: IR spectra of the amide I region of the pVII-mutant-M13 and the CNF-pVII-mutant-M13.


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Modified Filamentous Bacteriophage as a Scaffold for Carbon Nanofiber

Katarzyna Szot-Karpińska1,2*, Piotr Golec3, Adam Leśniewski1, Barbara Pałys4, Frank Marken5, Joanna Niedziółka-Jönsson1, Grzegorz Węgrzyn2, Marcin Łoś1,2

1

Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warszawa, Poland

2

Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland 3

Laboratory of Molecular Biology (affiliated with the University of Gdansk), Institute of

Biochemistry and Biophysics, Polish Academy of Sciences, Wita Stwosza 59, 80-308 Gdansk, Poland 4

Department of Chemistry, University of Warsaw, Pastuera 1 02-093 Warsaw, Poland 5

Department of Chemistry, University of Bath, Bath BA2 7AY, United Kingdom

*

To whom correspondence should be addressed: E-mail: [email protected]. Tel: +48 22 343 31 30, fax: +48 22 343 33 33

1



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ABSTRACT With the advent of nanotechnology, carbon nanomaterials such as carbon nanofibers (CNF) have aroused substantial interest in various research fields including energy storage and sensing. Further improvement of their properties might be achieved via application of viral particles such as bacteriophages. In this report we present a filamentous M13 bacteriophage, with a point mutation in gene VII (pVII-mutant-M13), that selectively binds to the carbon nanofibers to form 3D structures. The phage-display technique was utilized for the selection of the pVII-mutant-M13 phage from the phage display peptide library. The properties of this phage make it a prospective candidate for a scaffold material for CNFs. The results for binding of CNF by mutant phage were compared with those for maternal bacteriophage (pVII-M13). The efficiency of binding between pVII-mutant-M13 and CNF is about two orders of magnitude higher compared to the pVII-M13. Binding affinity between the pVIImutant-M13 and CNF was also characterized using atomic force microscopy, scanning electron microscopy and transmission electron microscopy, which confirmed the specificity of the interaction of the phage pVII-mutant-M13 and the CNF − the binding occurs via phage's ending where the mutated pVII protein is located. No similar behaviour has been observed for other carbon nanomaterials such as: graphite, reduced graphene oxide, single-walled carbon nanotubes and multi-walled carbon nanotubes. Infrared spectra confirmed differences in the interaction with CNF between the pVII-mutant-M13 and pVII-M13. Basing on conducted research we hypothesize that the interactions are non-covalent in nature, with π-π interactions playing the dominant role. Herein the new bioconjugate material is introduced.

Keywords: pVII protein, M13 bacteriophage, phage-binding, hybrid nanomaterials, carbon nanomaterials, carbon nanofibers

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INTRODUCTION Carbon nanofibers (CNF), with diameters in the range of 50-150 nm have found wide application in electrochemistry, as novel electrode material

1-3

, due to their unique properties

such as great electrical conductivity, high porosity and their ability to create well developed surfaces. Electrodes modified with such materials can be used to construct electrochemical sensors

4

or energy conversion

5, 6

and storage devices 7. CNF based electrodes can also be

used as a support for immobilization of biomolecules e.g. DNA

8, 9

. CNFs, apart from being

utilized for electrode modification, are also applicable as a hydrogen storage materials biomaterials for construction of medical devices such as implants

10

or

11

. Bearing in mind the

economic aspect, application of CNF in the industry is beneficial because their production is cheap and relatively straightforward 1. In order to expand industrial CNF applications it is highly desired to further improve aforementioned material electrical properties through developing its active surface. Recently some types of viruses, especially filamentous bacteriophages, have gained interest as carbon nanomaterials enhancing properties agents 12-16. Bacteriophages (phages) are bacterial viruses which are widely distributed in the environment. The estimated phages’ number on Earth is 1030 to 1032 17. They can take various shapes, from simple linear form (filamentous – M13, fd, f1), spherical (MS2), to more complex − like head-tail − structures (T4, T7)

18

. Filamentous phages have been used as an

additional nanometric-sized building blocks for creating new materials

12, 17, 19, 20

, owing to

their unique properties, such as ability to take shape of a long and thin threads, selforganization, and possibility of modification of their genetic material. The generation of such new phage-based materials is based upon the ability of constructing phages (e.g. M13) exposing peptide(s) specifically binding to a particular material of interest

20

. Interactions

between the phage and the material enable to produce hierarchical, self-assembled one-, twoand three-dimensional structures generation electrical

13, 23

12, 21, 22

, electronic

. These materials can be used for making next-

14, 24

, electrochemical 16, 25-27, biomedical

and other bio-/nanotechnological devices

19, 28

, optical

29

12, 14, 30

. Therefore the ability to create such hybrid

materials – bionanomatrials (phage-based materials) - might be of great interest for all those domains. Recent studies have shown that filamentous bacteriophages exposing selected peptides can bind specifically to various nanomaterials e.g. metal

29, 31, 32

and metal oxides 3



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nanoparticles 23, 33-35, semiconductors 24, 36, 37, perovskite 38, polymers 39, hydroxyapatite 40, or carbon nanomaterials. Among the latter, it has been demonstrated that genetically changed filamentous bacteriophages can be made to bind specifically to single-walled carbon 14, 15, 41, 42

, carbon planchet 43, highly-oriented pyrolitic graphite 43, graphene

nanotubes

13, 44,

, graphene-oxide 46, fullerene 47, or carbon nanohorns 48.

45

M13 bacteriophage is a filamentous phage with diameter of ca 6.5 nm and length of ca 880 nm. Its length can be modified and depends on the length of the packed single-strand DNA

17, 49

. The coat of the M13 bacteriophage is composed of roughly 2800 copies of the

major coat protein VIII (pVIII) stacked in units of 5 in a helical array and 5 copies of smaller coat proteins pIII and pVI or pIX and pVII, which are located at respective ends of the phage

17, 49

. The affinity of the phage towards target material (as well as its physicochemical

properties) depends on the structure of the peptide exposed on the virion's capsid. Properties of those peptides can be altered by fusing nucleic acids sequences through genetic modifications. Every capsid protein can be modified as a result of genetic engineering operations but pIII and pVIII are the most widely modified proteins of the M13 phage. However, too much intrusion into the pIII protein structure, e.g. insertion of too many amino acids (above 50), can diminish or inhibit adsorption of the phage on the bacterial cells leading to disruption of phage infection. Insertion mutations of the pIII of M13 phage, which are based on the addition of fewer than 50 amino acids (aa), are most widely described in the literature. These mutations have been applied for the construction of phages that bind a target material

32, 33, 39, 46, 47

. However even such relatively minor modification can reduce the

efficiency of phages' lifecycle or even stop it completely 50. Proteins pVII and pIX can also be modified. However, due to limited number of amino acids (a few aa) 51, which can be inserted into their genes', their application is not as often as pIII and pVIII. So far, these modified proteins have been used for binding antibodies

51-55

56, 57

and proteins

the initiation of progeny virions assembly and their stability

. pVII and pIX assist in

52

. Thus, a disruption of their

structures, by insertion of longer motives than a few aa, might impair these processes. Moreover, amino acids additionally inserted into the of pIII or pVII proteins may disturb their spatial conformation in such a manner that these amino acids will be removed through the viral protease based system responsible for recognizing unnatural amino acids appearing in the pIII or pVII protein. If such removal occurs the peptide (and the phage as well) will lose its intended function. Therefore, it is highly desirable to find a phage which exhibits a specific affinity to a target material without losing its infectious activity and stability. 4


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In this report a M13 bacteriophage with single point mutation in the gene coding the pVII protein (pVII-mutant-M13) is presented. It specifically binds to a carbon nanofibers but not to the other studied carbon based nanomaterials. To the best of our knowledge this is the first time where an M13 mutant with point mutation in the gene of the pVII protein (gpVII) was selected from phage peptide library through the phage display technique, and was used for specific binding of carbon nanofibers. The point mutation might have some impact on protein’s spatial conformation because a new amino acid − arginine − has extra positive charge comparing to glutamine

58

. However it preserves all of its important functions in

phage. Moreover the lifecycle of the phage with this mutation occurs with the same efficiency as for the maternal phage. The binding efficiency between the carbon nanofibers and the M13 phage with the wild-type pVII protein (marked as pVII-M13) is about two orders of magnitude lower compared to the M13 phage containing modified pVII protein (pVII with the point mutation). Moreover, the pVII-mutant-M13 does not exhibit any specific affinity towards other carbon nanomaterials such as: carbon nanoparticles, graphite, reduced graphene oxide, single-walled carbon nanotubes and multi-walled carbon nanotubes.

RESULTS AND DISCUSSION Selection and characterization of bacteriophage for carbon nanomaterials-binding The pVII-mutant-M13 bacteriophage has been identified using phage display technique utilized in hope of selecting bacteriophages' clones with affinity towards carbon nanoparticles (CNP). For detailed description of the procedure utilized CNP as a target in the panning procedure (see details in Materials and Methods). After third cycle of the panning procedure

49

, we chose 20 isolated clones to analyse

CNP-binding efficiency (O/I − a ratio of the output phage number (phages eluted/O) to the input phage number (phages incubated/I with target material, CNP)) (Fig. 1). The M13 clone which exhibited the highest value of the binding efficiency was selected for further detailed studies of its genome. First, we sequenced its genome region containing gene III (gpIII). For reason that no changes in gpIII were observed, the whole genome of pVII-mutant-M13 was sequenced. This analysis showed that selected clone exhibited a missense point mutation, single nucleotide change, in the gene of the pVII protein (gpVII) comparing to wild-type pVII protein (pVII-M13) (Table 1). The adenine residue was replaced with guanine at the position 1142. This mutation causes replacement of the glutamine residue with arginine at position 381 5



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in the protein (Gln381Arg). The details of the sequence of the modified gene can be seen in Supplementary Fig. F1. We suppose that this mutation might have some impact on protein’s spatial conformation. New amino acid − arginine − has extra positive charge comparing to glutamine 58. Therefore, the pVII protein might fold differently and its properties might diverge from unmodified protein. The clone with the point mutation (pVII-mutant-M13), and which revealed the highest affinity towards CNP was used for further studies. As a control, we used the M13 phage with the wild-type pVII protein (pVII-M13).

Table 1. Comparison of changed region of pVII (nucleotide and amino acid sequences) of the unmodified phage (pVII-M13) and modified phage with point mutation (pVII-mutant-M13).

Fig. 1a shows that there is no statistically significant difference between values of the binding efficiencies obtained for phage pVII-mutant-M13 and pVII-M13 with CNP. This suggests that the CNP are bound by pVII-mutant-M13 and pVII-M13 with almost the same binding efficiency (Fig. 1a, Table 2) indicating lack of the specific interactions between the pVII-mutant-M13 and studied material.

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Fig. 1. Efficiency of (a) CNP and (b) CNF binding by the phage (pVII-mutant-M13) with point mutation on the pVII protein and by the unmodified phage (pVII-M13). The presented results are average values from four experiments with RSD represented by error bars.

Additionally, the binding efficiency of the selected phage (pVII-mutant-M13) was tested for others available carbon nanomaterials such as: graphite (Gr), reduced graphene oxide (rGO), carbon nanofibers (CNF), single-walled carbon nanotubes (SWCNT), multi-walled carbon nanotubes (MWCNT). Surprisingly, the obtained results have shown the highest O/I value, which is equal to 2.2 x 10-3 for n=4 (RSD 6.9 x 10-4), when the CNF was used (Fig. 1b, Table 2). In order to check the selectivity of this binding phenomenon, the same volumes with the same concentration of the unmodified phage (pVII-M13) (8.4x1010 plaque-forming unit pfu/ml) was added to a freshly prepared CNFs suspension (see details in Materials and Methods section). The calculated O/I ratio is about two orders of magnitude lower compared to the one obtained for M13 phage with modified pVII protein (Fig. 1b, Table 2). The obtained value of O/I ratio indicates that the protein pVII of the M13 phage, with point mutation, specifically and strongly binds the carbon nanofibers. Moreover, that mutation does not affect infectivity of the pVII-mutant-M13. Its concentration changed only by ca. 16% after four rounds of amplification (3.8 x 1012 pfu/ml ± 6.2 x 1011 pfu/ml), simultaneously retaining its high affinity to bind CNFs (Table 2). Therefore the selected phage can be applied for modifying CNF-based materials.

Table 2 The average values of the calculated binding efficiency (O/I − ratio of the output phage number (phages eluted/O) to the input phage number (phages incubated/I with target material)) obtained for studied carbon nanomaterials and the pVII-mutant-M13 phage vs. pVII-M13. The presented results are average values from four experiments with RSD represented by error bars.

7



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Moreover, the obtained values of the binding efficiency for the other studied carbon nanomaterials and pVII-mutant-M13 are at least one, two or even four orders of magnitude smaller than that obtained for CNF (Table 2). This confirms that the binding between the pVII-mutant-M13 with mutation in gpVII and carbon nanomaterials other than CNF is very weak or does not occur at all. In contrast, when the lysate of the phage with unmodified protein pVII (pVII-M13) - the same volume and same concentration like it has been done for CNF - has been added to freshly prepared suspensions of studied carbon nanomaterials (Gr, rGO, SWCNTs, MWCNTs) in TBST. It has been noticed that the calculated average values of the binding efficiency are comparable with those obtained for modified protein pVII-mutantM13 (Table 2). This result shows that in the case of the studied carbon nanomaterials there is a lack of any specific binding by the pVII-mutant-M13 as well as by pVII-M13 because their values of the binding efficiency are comparable. Moreover, the values obtained for the carbon nanomaterials (Gr, rGO, SWCNTs) are very much the same as values obtained for the binding between pVII-M13 and the CNF or even five orders of magnitude smaller (MWCNTs). In these studies the bacteriophages displaying random 12-mer peptides fused to a minor coat protein (pIII) of M13 have been used. However in our case the phage with modified pVII and unmodified pIII has been selected by panning procedure. Such an unexpected result has also been demonstrated by Dong et. al 59. They have isolated a bacteriophage from a phage peptide library that display a linear 7-mer peptide on the pIII protein of M13 phage, which binds strongly to sulfur particles. But the binding site was along the phage virion (on pVIII protein), not at the end, where pIII protein is located 59. Microscopic characterization of bacteriophage carbon nanomaterials-binding

8


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The results of pVII-mutant-M13-CNF binding phenomenon were also confirmed by atomic force microscopy (AFM) (see details in Materials and Methods section) (Fig. 2). It is clearly seen that the numerous filamentous M13 phages with the modified protein pVII are present and specifically attached (with their endings where the pVII is located) to carbon nanofibers (Fig. 2a). These objects, and specific interactions between them, are also clearly visible in magnification (Fig. 2b). In the case of the phage with unmodified protein pVII (control experiment), these interactions are not present, and only bacteriophages separated from CNF aggregates are visible (Fig. 2c, d). The size of the depicted phages is in good agreement with the literature value 60.

Fig. 2 AFM images of CNF (a, b) specifically bound to the modified protein of the pVIImutant-M13 and (c, d) unbound with the unmodified protein of the pVII-M13 (c,d). AFM (a) amplitude error and (b-d) topographic images. Due to large difference between scales of phages and CNF particles, the above images contain only fragments of CNF particles. 9



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Also the images from transmission electron microscopy (TEM) confirm the presence of the specific binding of the pVII-mutant-M13 with CNFs (Fig. 3). Clusters and ribbons of selforganized pVII-mutant-M13 phages fibers specifically attached to carbon nanofibers are visible (Fig. 3a, b). The recorded fibers of the modified pVII-mutant-M13 phages are connecting carbon nanofibers leading to formation of a scaffold for them. Such alignment of structures was not observed in the case when M13 phage with unmodified protein pVII was added to the suspension of carbon nanofibers (Fig. 3c, d). Unfortunately the structure of the pVII has not been solved and its arrangement in the M13 particle has not been determined 18. Thus, it is hard to predict the structural conformation of the mutated protein and the origin of its interaction with CNF.

Fig. 3 TEM images of CNF (a, b) specifically bound with the pVII-mutant-M13 and (c, d) with the unmodified pVII-M13 bacteriophages visibly unbound. In order to gain insight into the nature of these interactions we used UV-Vis spectroscopy, zeta potential and XPS analysis. Covalent bonding does not occur between the pVII-mutant-M13 and CNF. This was confirmed by XPS analysis which reveals no changes 10


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of the C1 peaks recorded for the pVII-mutant-M13 after addition of the CNF in comparison with the pure pVII-mutant-M13 (Supplementary Fig. F2). The specific binding between the pVII-mutant-M13 and the CNF might be explained as a result of interactions of the modified pVII protein with the CNF through π-π binding with the aromatic residue of phenylalanine or tyrosine, which are near to the region where the modification occurs in the protein pVII, and which could have been made possible by changes in the conformation of the protein resulting from replacement of the glutamine with arginine. This hypothesis is in agreement with the result obtained from the UV-Vis absorption spectra as these results clearly indicate π-π interactions Fig. 4. The spectrum of the pVII-mutant-M13 (Fig. 4 pVII-mutant-M13 black line) was compared with the spectrum of the pVII-mutant-M13 with CNF from which the spectrum recorded for clean CNF solution was subtracted (Fig. 4 CNF-pVII-mutant-M13-CNF blue line). One can observe that the peak about 230 nm has shifted towards lower wavenumber. This result support the hypothesis that binding of the pVII-mutant-M13 with the CNF occurs through π-π interactions 61, 62 (Fig. 4). No shifting of the peak has been observed when pVIIM13 lysate was added to the CNF solution (not shown).

Fig. 4 Absorbance spectra for pVII-mutant-M13 solution (black line) and CNF-pVII-mutantM13-CNF (blue line). The shift of the peak at about 230 nm marked with red area. The presence of the arginine instead of the glutamine in the pVII of the pVII-mutant-M13 phage results in extra positive charge which is also confirmed by the zeta potential values. These values show that the pVII-mutant-M13 (ζ = -10.4 ± 1.4 mV) is less negatively charged than the pVII-M13 (ζ = -18.6 ± 0.5 mV). Moreover, an enrichment of the pVII-mutant-M13 with a nitrogen content, due to the presence of the amine amino acid - arginine, has been 11



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confirmed by XPS analysis. The result shows that content of the nitrogen for the pVII-mutantM13 is 10.24 %, whereas for the pVII-M13 equals 8.80 %. We assume also that the extra positive charge might have some effect on protein’s spatial conformation resulting in electrostatic interactions with positively charged surface of CNF (ζ = + 21.3 ± 1.31 mV) than with negatively charged CNP (ζ = -34.8 ± 1.08 mV). Nonetheless, the electrostatic interactions do not play a crucial role in binding pVIImutant-M13 with CNF. Small values of the binding efficiency obtained for other negatively charged carbon nanomaterials, such as graphene oxide (1.86 x 10-5 ± 6.56 x 10-6) and carboxyl-functionalized SWCNTs (2.92 x 10-5 ± 5.33 x 10-6) (Supplementary Table 1), confirm that the pVII-mutant-M13 does not exhibit affinity towards other negatively charged materials. In order to get further insight into the nature of the interaction between pVIImutant-M13 and studied materials, we calculated the binding efficiency for positively charged carbon nanopartciles (sulfonamide-functionalised CNP). Surprisingly the obtained value equals zero indicating a lack of any specific binding by the pVII-mutant-M13 (Supplementary Table 1). Additionally, the suggested changing of the pVII protein spatial conformation (pVIImutant-M13) might be resulting in stronger hydrophobic properties of the affected area of the modified pVII protein than unmodified one making interactions (in aqueous environment) between corresponding area of phage's surface and the CNF more probable to occur and producing more stable binding. The specific binding of the pVII-mutant-M13 has not been observed for other carbon nanomaterials such as: reduced graphene oxide, single-walled carbon nanotubes, multi-walled carbon nanotubes, whereas very weak binding has been observed for graphite. Consistent results have been obtained on the basis of the calculated value of the binding efficiency (Table. 2) and images obtained from TEM (Fig. 5). In the TEM image made for graphite suspension and M13 phage with modified protein aggregates of phages fibers, which interact with the material in a manner different then observed for the CNF, can be noted (Fig. 5a). This behaviour of the M13 phage particles - a natural tendency towards aggregates formation - has been already reported by Sanders et.al.

63

. In the case of samples where the carbon

nanotubes are present, phage’s fibers with modified protein pVII-mutant-M13 are also clearly visible. But they don't group into aggregates and exhibit no specific interactions between the studied materials and their endings (Fig. 5c, d). In the case of reduced graphene oxide image

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show lack of any phage particles confirming lack of binding interactions between the studied materials.

Fig. 5 TEM images of carbon nanomaterials (a) graphite (Gr), (b) reduced graphene oxide (rGO), (c) single-walled carbon nanotubes (SWCNTs), (d) multi-walled carbon nanotubes (MWCNTs) and their interactions with the pVII-mutant-M13 phages. The surface of the CNF-pVII-mutant-M13 and CNF-pVII-M13 materials was also investigated by scanning electron microscopy (SEM) (Fig. 6). SEM images revealed significant differences in the surface morphology of the CNF-pVII-mutant-M13, CNF and CNF-pVII-M13 materials. The CNF-pVII-mutant-M13 material forms aggregates of the size from few hundreds nanometers to few micrometers (Fig. 6b), whereas the array of CNF and CNF-pVII-M13 is more uniform and porous (Fig. 6a, c). The presence of the aggregates indicates that the pVII-mutant-M13 phages bind CNF. However, it is difficult to judge whether the pVII-mutant-M13 phages bind specifically, with their ends, to CNF. The lack of the aggregates confirms that the pVII-M13 phages do not interact with CNF and morphology of this material is similar to bare CNF.

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Fig. 6 SEM images of CNF (a) with the pVII-mutant-M13 (b) and the pVII-M13 (c) materials deposited on the ITO electrode. Spectroscopic characterization of the bacteriophage CNF-binding The interactions of the pVII-mutant-M13 with CNF was confirmed by infrared spectra (IR) microscopy in the reflectance mode (Fig. 7). The spectrum of the CNF-pVII-mutant-M13 deposited on the silicon wafer was compared with the modified phage without CNF (Fig. 7 pVII-mutant-M13) and unmodified phage with CNF (Fig. 7 CNF-pVII-M13). At each of these spectra the protein (C=O) amide I band is found at ca 1650 cm−1 and amide II at ca 1550 cm−1. However, the most prominent change introduced by the CNF-pVII-mutant-M13 is a split band from amide I. The amide I band (1600-1700 cm-1) is useful for studying the secondary structural composition and structural dynamics of proteins so any changes in this band might indicate some disturbance in protein's spatial conformation 64, 65. Obtained results suggest that spatial conformation of one of the protein of the pVII-mutant-M13 - probably the modified pVII - is changing when it interacts with CNF. Also it confirms that pVII-mutantM13 interacts with CNF in a different manner than pVII-M13. The split of the amide I band suggests further that the part of the protein interacts with CNF, while some pVII molecules remain unbound. The remaining bands result probably from other protein motions, like amide II band around 1520 cm-1, amide III at 1466 cm-1, C-O stretching at 1050 cm-1. Some minor bands between 1000 and 1500 cm-1 might correspond to the protein side groups. The bands due to possible surface functionalities of CNF are difficult to discern, as visible in the spectrum of the unmodified CNF (Fig. 7). The very broad overlapping bands ranging from 2800 to 3600 cm-1 indicate presence of O-H and N-H groups involved in hydrogen bonds. The diminishing of the overall intensity and formation of a narrow band at 3182 cm-1 suggests that a part of the hydrogen bonding network is disturbed by the interaction between pVII-mutant-M13 and CNF (Fig. 7 CNF14


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pVII-mutant-M13). Such alteration in the hydrogen bonding network is consistent with the changes of the protein spatial conformation. The relatively small intensity of the -OH stretching region is observed for the unmodified phage bound to CNFs (the CNF-pVII-M13 spectrum). This observation might confirm that fewer unmodified bacteriophages are bound to CNFs comparing to the mutant phage.

Fig. 7 Infrared spectra of CNF with the pVII-M13 (CNF-pVII-M13), pVII-mutant-M13 (CNF-pVII-mutant-M13) and the pVII-mutant-M13 without CNF material deposited on the on silica surface. Inset: IR spectra of the amide I region of the pVII-mutant-M13 and the CNF-pVII-mutant-M13. CONCLUSIONS In this study, the filamentous M13 bacteriophage with a single point mutation in the gpVII (pVII-mutant-M13) was selected through the phage-display technique. The identified phage specifically binds carbon nanofibers - via its end where the pVII protein is located. The binding occurs mainly through non-covalent interactions; electrostatic and π-π, but the latter one seems to be dominating in binding the pVII-mutant-M13 with CNFs. The binding efficiency between the carbon nanofibers and the M13 phage with the modified pVII protein is about two orders of magnitude higher compared to the M13 phage with unmodified pVII 15



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protein. The mutated bacteriophage maintains its infectivity and stability. The discovery of this ability of CNF binding by mutated M13 with the phages' natural tendency to aggregate could enable the development of novel functional hybrid CNF-based materials using the described M13 mutant virions as a scaffold or platform for energy storage and sensing. For example the presented material (pVII-mutant-M13-CNF) can be used to form a highly developed network via layer by layer method, similarly like it was presented by Belcher

66

.

The low-cost and high scalability of the phage amplification process is an additional advantage of described approach. MATERIALS AND METHODS Chemicals and Materials Negatively charged carbon nanoparticles (CNP) (ca. 7.8 nm mean diameter, with a typical bulk density of 320 g dm-3, Emperor 2000) were supplied by Cabot Corporation (Dukinfield, United Kingdom). These nanoparticles were used for preparation of positively charged CNP (sulfonamide-functionalised CNP) following a procedure described earlier

67

. Graphite

powder MP-300 (Gr), with diameter ca 25 µm, was purchased from Carbon IndustrieProdukte GMBH, single-walled carbon nanotubes (SWCNTs) (2 nm diameter and ca. 1 µm in length) were from Thomas Swan & Co (UK), and multi-walled carbon nanotubes (MWCNTs) (20-30 nm diameter and 0.5-200 µm in length) from Sigma-Aldrich. Carboxyl-functionalized SWCNTs were purchased from Dropsens (2 nm diameter and length of several microns). Graphene oxide (GO) and reduced graphene oxide (rGO) were prepared according literature procedures

68, 69

, respectively. Carbon nanofibers (CNF), with diameters in the range 10-500

nm, were grown from an ethylene/hydrogen mixture in contact with an iron catalyst following a literature procedure 1. ITO coated glass (resistivity 8–12 Ω/ square) was from Delta Technologies. A phages that display a linear 12-mer peptide on the pIII protein of M13 KE phage (Ph.D.-12 Phage Display Peptide Library Kit, New England Biolabs, NEB). Tween 20, Trisma base, glicyne, bovine were purchased from Sigma-Aldrich and used without any further purification. In the studies the aqueous solutions of carbon nanomaterials were used. The surface of the used carbon nanomaterials was not functionalized, except the carboxylfunctionalized SWCNTs, negatively and positively charged CNP.

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Selection and characterization of bacteriophage carbon nanomaterials-binding. Panning procedure was performed according to the Phage Display Manual (NEB)

49

. At the

beginning the CNP were utilized as a target for phage display technique. Briefly, 10 mg of CNP powder were washed six times with the TBST buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.6 % Tween-20) to remove any carbon nanoparticles that do not sediment during centrifugation at 10,000 × g for 2 min (which would negatively interfere with phage isolation at later steps of the procedure) and to establish the equilibrium of buffer conditions of the mixture (which is crucial for effective binding of phages to any surface). 10 µl of phage peptide library was incubated with 1 mL CNP in the TBST buffer (at final CNP concentration of 10 mg/mL) for 1 h at room temperature (RT). This incubation time was chosen on the basis of results of preliminary experiments, in which shorter incubation was less efficient in isolating carbon nanoparticles-binding phages, while extending the time over 1h did not change the results significantly. Unbound phages were then separated from CNP-binding phages by centrifugation (10,000 × g, 1 min, RT) and were subsequently removed by decanting the supernatant. The pellets containing CNP with bound phages were washed ten times with the TBST buffer. Phages which bound CNP were eluted with 1 mL of 0.2 M glycine– HCl (pH 2.2) for 10 min, and finally neutralized with 150 µL of 1 M Tris–HCl (pH 9.1). Phages were then multiplied on Escherichia coli ER2738 (NEB) according to the NEB protocol and used in the next panning procedure (briefly, for phage multiplication, 0.2 mL of an overnight E. coli ER2738 culture were mixed with eluted phages, transferred to 20 mL of fresh LB medium in a 250 mL flask, and incubated with shaking (160 rpm) at 37 °C for 5 h). Three rounds of panning procedure were performed. Following the last panning, the eluted phages were titrated on agar plates supplemented with IPTG/XGal as previously described 70. 20 clones of phages from individual plaques were amplified as described above, and their DNAs were isolated and purified according to Wilson

71

and sequenced commercially using

the 96 gpIII sequencing primer (NEB) or primers which were designed by us for sequencing whole M13 genome (5'-CCAATAAAATCATACTACAGGCAAGGC-3', 5'-GTGGGAACAAACGGCGGATTG3', 5'-AGTGAGACGGGCAACAGCTG-3', 5'-TCAGTGAGGCCACCGAGT-3', 5'-TAGATTAGAGCCGTCAATAG-3',5'-CCTTGAAAACATAGCGATAGC-3', 5'CCAAGAACGGGTATTAAACC-3', 5'-GCCGAACAAAGTTACCAG-3', 5'-AGAACCACCACCAGAGCCGC-3', 5'-CATAAGGGAACCGAACTGACC-3', 5'-GCCAGAGGGGGTAATAGT-3' and 5'-GACAGCCCTCATAGTTAGCG-3'). 17



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One of the 20 clones was utilized for further studies and it is marked as pVII-mutant-M13 (M13 phage with point mutation in gene of the protein pVII). As a control the M13KE phage (NEB) with the wild-type pVII protein was used and marked as pVII-M13. The panning procedure was repeated in the same way for all studied carbon nanomaterials with pVII-mutant-M13 and pVII-M13, separately. In order to calculate the value of the binding efficiency for studied carbon nanomaterials to freshly prepared suspensions of TBST with various carbon nanomaterials (Gr, rGO, GO, CNF, SWCNT, carboxyl-functionalized SWCNT, MWCNT, positively charged CNP) the same volume (10 µl) with the same concentration (3.8 x 1012 pfu/ml ± 6.2 x 1011 pfu/ml) of the phage (pVIImutant-M13) lysate was added, like it has been done in the case of CNP. Eluted phages from the corresponding carbon nanomaterials suspensions have been titrated and the average values of the binding efficiency have been calculated. In the same way the value of the binding efficiency was calculated for unmodified phage (pVII-M13). So the same volume (10 µl) and same concentration 8.4x1010 pfu/ml of the unmodified phage (pVII-M13) lysate was added to the freshly prepared suspensions of TBST with the studied carbon nanomaterials, separately, like it has been done in the case of CNPs. AFM analysis Aliquot of CNF suspension (10 mg/ml) in toluene were dropped onto freshly prepared mica surface (cleaved mica). After solvent evaporation aliquot of bacteriophage M13 suspension (5 µl) with the protein pVII-mutant-M13 or unmodified protein pVII-M13 was dropped and left for adsorption (30 min) in closed space in order to avoid water evaporation. After adsorption, samples were rinsed with water, dried under argon flow and then were investigated with AFM in tapping mode. Infrared spectroscopy Samples were prepared in a following way: 5 µl droplet of each suspension: CNF, CNF with pVII-mutant-M13 and CNF with pVII-M13 have been deposited on the silicon wafer surface and water was allowed to evaporate in an ambient conditions. The sampled area for a single spectrum was equal to 100 µm2. The spectral resolution was equal to 4 cm−1 and typically 64 scans were averaged for a single spectrum. Bare silicon wafer was used to record the background.

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SEM analysis Samples were prepared in a following way: 5 µl droplet of each suspension: CNF, CNF with pVII-mutant-M13 and CNF with pVII-M13 have been deposited on the ITO surface and water was allowed to evaporate in an ambient conditions. TEM analysis M13 phages lysates in suspensions of TBST with various carbon nanomaterials (carbon nanoparticles, carbon nanofibers, graphite, reduced graphene oxide, single-walled carbon nanotubes and multi-walled carbon nanotubes) with modified and unmodified protein pVIImutant-M13 and pVII-M13, respectively - were adsorbed onto carbon-coated grids (Sigma) for 3 minutes, stained with 1.5% uracyl acetate, and examined using transmission electron microscope (TEM) Philips CM100. The studied phages and carbon nanomaterials were photographed by iTEM program. UV-Vis analysis 0.5 ml of distillated water was placed in UV-Vis cuvettes. Next: 500 µl of pVII-mutant-M13 lysate, 500 µl of pVII-M13 lysate, CNF (3mg/ml), 500 µl of pVII-mutant-M13 lysate with CNF (3mg/ml) and 500 µl of pVII- M13 lysate with CNF (3mg/ml) have been added to the system, separately. For each solution a UV-Vis spectrum was recorded. The spectrum recorded for clean CNF solution was subtracted from the spectra recorded for the CNF with pVII-mutant-M13 and CNF with pVII-M13 scans. The obtained spectra has been labeled CNF-pVII-mutant-M13-CNF. Absorbance changes were monitored at 230 nm (π-π* transition absorption band). UV absorption spectra were recorded by Evolution 300 UV−vis spectrophotometer, Thermo Scientific. All measurements were performed at room temperature (22 ± 2 °C). X-ray photoelectron spectroscopy (XPS) Samples were prepared in a following way: 5 µl droplet of phages lysates: pVII-mutant-M13 and pVII-M13 have been deposited on the silicon wafer surface and water was allowed to evaporate in ambient conditions. X-ray photoelectron spectroscopy (XPS) experiments were performed in a PHl 5000 VersaProbe - Scanning ESCA Microprobe (ULVAC-PHI, Japan/USA) instrument at a base pressure below 5×10-9 mbar. Monochromatic AlKα radiation was used and the X-ray beam, focused to a diameter of 100 µm, was scanned on a 250×250 µm surface, at an operating power of 25 W (15 kV). Photoelectron survey spectra were 19



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acquired using a hemispherical analyzer at pass energy of 117.4 eV with a 0.4 eV energy step. Core-level spectra were acquired at pass energy of 23.5 eV with a 0.1 eV energy step. All spectra were acquired with 90° between X-ray source and analyzer and with the use of low energy electrons and low energy argon ions for charge neutralization. After subtraction of the Shirley-type background, the core-level spectra were decomposed into their components with mixed Gaussian-Lorentzian (30:70) shape lines using the CasaXPS software. Quantification calculations were conducted with using sensitivity factors supplied by PHI. Apparatus AFM images were obtained with a MultiMode AFM instrument using a Nanoscope V controller (Bruker) operating in tapping mode. The images were processed with Gwyddion 2.32 SPM data visualization analysis tool. Infrared spectra were recorded using the Nicolet iN10-MX-FTIR microscope (Thermo Scientific). The experiments were carried out using the reflectance mode and the MCT detector. SEM images were taken with a FEI Nova NanoSEM 450 scanning electron microscope. TEM images were taken with a FEI Nova NanoSEM 450 scanning electron microscope. Philips CM100. Phages were photographed by iTEM program. The zeta potential was measured using the Malvern Instruments. UV absorption spectra were recorded by Evolution 300 UV−vis spectrophotometer, Thermo Scientific. XPS experiments were performed in a PHl 5000 VersaProbe - Scanning ESCA Microprobe (ULVAC-PHI, Japan/USA) instrument at a base pressure below 5×10-9 mbar. ASSOCIATED CONTENT Supporting Information The details of the sequence of the modified gene. Table of the average values of the calculated binding efficiency obtained for studied carbon nanomaterials and C1s XPS spectra of the pVII-mutant-M13 and the phage with CNF (pVII-mutant-M13-CNF). This material is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: +48 22 343 31 30, fax: +48 22 343 33 33. Notes The authors declare no competing financial interest. 20


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ACKNOWLEDGEMENT This work was funded by the Polish National Science Centre via a FUGA grant (post-doctoral internship UMO-2012/04/S/NZ1/00039) to Dr. Katarzyna Szot-Karpińska. JNJ and AL thank the Foundation for Polish Science under the FOCUS Programme/Grants 3/2010/Grants. KSK gratefully acknowledge MSc. Eng. Marcin Karpiński for fruitful discussions.

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Graphical abstract

Schematic illustration of carbon nanofibers (CNF) binding by the M13 phage with point mutation in pVII protein (pVII-mutant-M13).

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Modified Filamentous Bacteriophage as a Scaffold ... - ACS Publications

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