Engineered Adhesion Peptides for Improved Silicon Adsorption

Subscriber access provided by University of Pennsylvania Libraries

Article

Engineered adhesion peptides for improved silicon adsorption Sathish Kumar Ramakrishnan, Saïd Jebors, Marta Martin, Thierry Cloitre, V. Agarwal, Ahmad Mehdi, Jean Martinez, Gilles Subra, and Csilla Gergely Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b02857 • Publication Date (Web): 06 Oct 2015 Downloaded from http://pubs.acs.org on October 10, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir 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.

Page 1 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Engineered adhesion peptides for improved silicon adsorption Sathish Kumar Ramakrishnan,a Said Jebors, b Marta Martin, a Thierry Cloitre,a Vivechana Agarwal,c Ahmad Mehdi,d Jean Martinez,b Gilles Subrab and Csilla Gergely a* a

Laboratoire Charles Coulomb (L2C), UMR 5221 CNRS-Université de Montpellier, Montpellier,

F-France.; bInstitut des Biomolécules Max Mousseron (IBMM), UMR5247 CNRS, Université de Montpellier, 15 Avenue Charles Flahault, Montpellier, France ; cICIICAP- Universidad Autonoma del Estado de Morelos, Av. Universidad 1001, Col Chamilpa, Cuernavaca, Morelos, México ;

d

Institut Charles Gerhardt - UMR5253, Université de Montpellier, F-34095,

Montpellier, France * Address correspondence to: [email protected]

KEYWORDS: Peptide-Semiconductor interactions, engineered peptides, Material specific peptides, biomaterials and peptide synthesis.

ACS Paragon Plus Environment

1

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 25

ABSTRACT

Engineering peptides that present selective recognition and high affinity towards a material is a major challenge for assembly-driven elaboration of complex systems with wide applications in the field of biomaterials, hard-tissue regeneration and functional materials for therapeutics. Peptide–material interactions are of vital importance in natural processes, but less exploited for the design of novel systems for practical applications due to poor understanding of mechanisms underlying these interactions. Here, we present an approach based on the synthesis of several truncated peptides issued from a silicon-specific peptide recovered via phage display technology. We use the photonic response provided by porous silicon microcavities to evaluate the binding efficiency of fourteen different peptide derivatives. We identify and engineer a short peptide sequence (SLVSHMQT) revealing the highest affinity towards p+-Si. The molecular recognition behavior of the obtained peptide fragment can be revealed through mutations enabling identification of the preferential affinity of certain amino acids towards silicon. These results constitute an advance in both the engineering of peptides that reveal recognition properties for silicon and the understanding of biomolecule-material interactions.


2

Page 3 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

INTRODUCTION

Peptides are the fundamental components of several biological processes; with unique structures and sequences, they form functional nano-assemblies leading to materials of complex shapes and with remarkable properties. For instance, sea shell,1 tooth,2 and bone3 are natural materials produced in a highly controlled manner, via biological self-assembling processes that are difficult to achieve in synthetic systems with similar phase compositions. Proteins play a major role in directing the assembly of biomaterials with precise control of phase composition resulting in complex architectures with multifunctional properties.4 Such natural biological structures provide valuable clues to the design of bio-inspired materials and various systems for biomedical applications.5,6 Living organisms express biomineralization polypeptides that have the ability to direct the synthesis and assembly of inorganic materials of a desired material composition and phase.7–9 However, such naturally occurring peptides are limited to a certain number of inorganic materials available in the biological systems that express them. There are many other materials of interest such as gold, platinum, and semiconductors that may not have a natural peptide to mediate their growth. Researchers now employ combinatorial biological techniques to select peptides that exhibit exceptional affinities towards materials for various applications; such as for controlling the nucleation of nanocrystals,10 inorganic nanoparticles assembly,11,12 and use as molecular linkers.13 Peptides bearing exquisite molecular recognition properties are well exploited to control the shape of nanocrystal growth.14,15 Understanding the mechanisms of underlying interactions between peptides and inorganic surfaces is essential for advancing towards rational tailoring of specific peptides with desired properties. Several


3

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 25

attempts were made in the past, experimentally,16,17 theoretically,18,19 or both,20,21 to understand key amino acid residues in the sequence and design of peptides by substituting, deleting, and rearranging them to enhance their affinity towards inorganic surfaces. Tomczak et al. used five different variants of the A3 (AYSSGAPPMPPF) gold affinity peptide, addressing mutation and scrambling of key residues within the sequence. They found that replacement of methionine with alanine (A3-A) exhibited the largest affinity for gold, while proline-substituted peptide (A3-P) showed no binding, and the other three-peptide variants displayed moderate binding affinities.16 Oren et al. used a bio-informatics approach to design novel peptides with predictable affinities towards quartz by generating new scoring matrices.20

Silicon- (Si-) binding peptides were reported as efficient molecular linkers for biomolecule adhesion, enabling a 21 times enhanced detection sensitivity compared to that achieved via chemically functionalized sensors.13 However, further progress in this domain is limited due to the lack of clear understanding of the adsorption mechanism and the role of individual amino acids in the peptides’ specific affinity for materials. Recognizing the key residues responsible for adhesion could provide optimal peptide sequences with increased binding properties towards materials greatly cutting down the cost of lengthy peptide synthesis. Indeed, several studies have addressed to identify the amino acids responsible for the binding affinity of peptides.14,22 The SPGLSLVSHMQT 12-mer peptide (named P peptide)13, recovered via the phage display technique, exhibits high binding affinity for the p+-Si surface, and is a good material linker for immobilization of biological molecules on this semiconductor. Consequently, given its competitive behavior from a pool of billions of peptides, P represents a good model sequence to study the role of the amino acids in its preferential adsorption towards p+-Si.


4

Page 5 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

In a previous work, we employed molecular dynamic calculations to explore the binding affinity of peptides on Si that are recovered by phage display technology against n-type Si.25 The key amino acids in the sequence of these peptides, responsible for their high affinity were identified by performing a simulated residual scanning analysis. Also, we found that any modification in the C-terminus of the peptide induced a significant decrease in binding affinity. The reason for such a behavior is still unknown. This significant affinity against a cross over surface arise a question about the origin of selective binding that we attempt to address in this work through an experimental approach. We designed truncated peptide analogues of the SPGLSLVSHMQT (peptide P), and assessed their adhesion to p+-Si against the one obtained with the original P peptide. The primary objective was to determine and engineer the shortest peptide sequence with the highest affinity towards silicon. The molecule’s binding event was measured as an optical response produced by porous silicon (PSi) microcavities that are basically 1-D photonic structures composed of an active layer placed between two Bragg reflectors.23 Due to their conveniently tunable structural and optical properties, PSi microcavities are widely used for biosensing applications. The fluorescence signals produced by the FITC labeled streptavidin captured by the microcavities functionalized with biotinylated peptides have been used to quantify the binding of various peptide sequences.


5

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 25

EXPERIMENTAL

Fabrication of Porous Silicon Microcavities All porous silicon photonic structures were prepared by electrochemical anodization of highly boron doped p+-type Si(100) wafers of 0.001–0.002 Ω cm resistivity in a 3:7:1 (v/v) hydrofluoric acid (HF, 48%), ethanol (EtOH, 98%) and glycerol (98%) electrolyte solution. A computer program was used to precisely control the anodization time and current density in order to generate high and low porosity layers with an optical thickness of one quarter of the wavelength, with the resonance wavelength in the range of 900 nm. PSi microcavity samples, with a configuration (HL)×5 HH (LH)×5 (with H/L being the high/low porosity layers), were fabricated by alternating a high anodization current pulse (75mAcm−2) for 6.4 s, followed by a short low current pulse (35mAcm−2) for 11.5s. The corresponding Bragg mirror consisted of 12 periods (HL)x12, to enhance the reflectivity. The microcavities were fabricated with the high porosity layer at the top to facilitate penetration of molecules.

Peptide Synthesis The reagents, amino acids and resins were purchased from Iris Biotech. Solvents were purchased from Fluka and Aldrich. Peptides were prepared using microwave assisted Fmoc SPPS (see supporting information),24 on Fmoc-Rink amide PS resin. The introduction of Biotin at the Cterminus of the peptide sequences was achieved by using Fmoc-Lys(biotin)-OH as building block which was prepared as follows: To a solution of 1g of Fmoc-Lys-OH DMF (100 mL) was added DIEA (2 eq, 130µL) and biotin preactivated as benzotriazole derivative (biotin-Bt, 1eq,


6

Page 7 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

714 mg). The reaction mixture was stirred for 30 minutes at room temperature. The solution was concentrated under vacuum. Fmoc-Lys(Biotin)-OH was precipitated with acetonitrile. The filtrate was washed with acetonitrile (3×), water (3×) and dried under vacuum. FmocLys(Biotin)-OH was analyzed by HPLC, LC/MS, 1H and 13C NMR (see supporting information) and used without further purification. The anchoring of Fmoc-Lys(Biotin)-OH (3 eq) on H-Rink amide resin was prepared as follows: To a DMSO suspension of deprotected H-Rink Amide PS resin was added DIEA (6 eq) and BOP (3 eq) in DMSO. The reaction mixture was stirred at room temperature for 2 h, then washed with DMSO/MeOH/DIEA (17/2/1) (3×), DCM (3×), MeOH and DCM.

Functionalization of the porous silicon Phosphate buffer saline Tween (PBS and PBST) was diluted tenfold in deionized water to minimize the salt crystallization while functionalizing the PSi devices. The silicon native oxides were removed by chemical etching, using HCl:H2O 1:10, for 10 min followed by deionized water rinse for a minute. The surfaces were then immediately incubated with 20 µM of synthesized biotinylated peptides (diluted in BSA 0.1% containing PBST/10) for 2 hours, followed by a thorough rinsing step in PBST/10 to remove unbound peptides. Then, the samples were incubated with a 10 µM solution of streptavidin conjugated fluorescein isothiocyanate (Sigma Aldrich, S3762), FITC, (in presence of BSA 0.1% in PBS/10) for 2 hours followed by three consecutive PBS/10 buffer rinses. All washing steps were performed with the buffer followed by deionized water to avoid salt crystal formation. After each functionalization step, the samples were gently dried under a stream of N2 before recording their reflectivity spectra


7

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 25

Reflectivity spectra Reflectivity spectra were measured by our custom built reflectometer. One end of the reflection probe (Ocean optics, QR400-7-VIS-NIR) is coupled to the halogen lamp source (Harvard Apparatus, IX 5000-1) and the other end to the spectrometer (Thorlabs, CCS100). A spectrum (resolution of 1- 2 nm) within the wavelength range 400-1050 nm was acquired after each surface modification by averaging 20 scans with an integration time of 10 ms.

Fluorescence microscopy Fluorescence images were recorded with a Nikon epifluorescence microscope (Eclipse TE 2000E) equipped with a Sony Nex-3 charge-coupled device (CCD) camera. The 10x Nikon PlanFluor (N.A. 0.30; W.D 15.2mm) microscope objective was used to collect the fluorescence emission of FITC (excitation 480 nm; emission 540 nm).

Fluorescence Spectra Fluorescence signals obtained after illuminating the PSi samples at a wavelength of 480 nm were recorded. To quantify fluorescence of different samples, the emitted light was collected by an optical fiber (Ocean optics, P1000-2-VIS-NIR) coupled to a spectrograph and a back-illuminated CCD (Princeton instruments, Pixis: 400).


8

Page 9 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

RESULTS AND DISCUSSION

Design of peptide variants Twelve different variants (Table 1) of the SPGLSLVSHMQT (P) peptide, previously elaborated by phage display against the p+-Si,

14

were synthesized. They are all truncated forms of the P

peptide, shortened by removing residues sequentially either at the N-terminus (PN1-PN5 peptides) or at the C-terminus (PC1-PC7 peptides). The peptides were modified at the Cterminus with the GGGSK pentapeptide serving as a spacer between the biotin and the peptide. GGGS is commonly used as a spacer to set apart a bioactive moiety from a functional element design, for example, to interact with a surface. It is described to be flexible thanks to the unsubstituted Glycine residues, in order to avoid unwanted orientation of bioactive moiety. It also includes a Serine residue, which gives the spacer some hydrophilicity. In our case, we added a lysine residue, which is not directly part of the spacer but is used to display the biotin moiety on the amine epsilon side chain. The biotinylated peptides were used to functionalize the PSi substrates to capture the fluorophore tagged streptavidin (Scheme 1).


9

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 25

Scheme 1: Supposed adsorption of biotinylated peptide analogues of P on PSi to capture fluorescent streptavidin. Note the P analogues (colored balls), the GGSK spacer and the biotin (yellow ball) where FITC labelled streptavidin binds.

Table 1. Truncated peptide sequences designed based on the peptide (P) SPGLSLVSHMQ, previously elaborated as a high affinity binder for p+-Si. The cavity resonance shifts of the PSi samples measured after peptide adsorption and capture of streptavidin are also presented. All the synthesized peptides were modified with a GGGSK(Biotin) linker (not shown in the table for clarity of the basic peptide sequences). aThe property values were calculated using pI/Mw tool at http://expasy.org.

Id

Peptide

PIa

∆λPeptide (nm)

∆λStrep (nm)

P PC1

-SPGLSLVSHMQT-

6.46

PGLSLVSHMQT

7.17

4.05 ± 0.10

2.31 ± 1.67

9.00 ± 0.54

15.00 ± 0.94

PC2

GLSLVSHMQT

6.74

2.51 ± 0.99

3.92 ± 0.3

PC3

LSLVSHMQT

6.74

2.26 ± 0.26

0.78 ± 0.08

PC4

SLVSHMQT

6.46

8.83 ± 0.29

14.6 ± 1.37

PC5

VSHMQT

6.71

7.87 ± 0.94

9.74 ± 2.12

PC6

SHMQT

6.46

3.62 ± 1.77

9.48 ± 0.98


10

Page 11 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

PC7

HMQT

6.74

2.17 ± 1.37

9.82 ± 1.76

PN1

SPGL

5.24

2.21 ± 0.44

-4.09 ± 0.97

PN2

SPGLSL

5.24

2.41 ± 1.46

-2.03 ± 0.38

PN3

SPGLSLVS

5.24

1.31 ± 1.50

-1.35 ± 0.43

PN4

SPGLSLVSHM

6.46

1.12 ± 0.13

-1.31 ± 0.63

PN5

SPGLSLVSHMQ

6.46

3.95 ± 1.01

5.22 ± 0.67

Peptide to Silicon Adhesion Measurements The photonic response of the PSi microcavities was used to quantify the adhesion of the peptide derivatives. The principle for the detection of molecules adsorbed onto porous silicon structures relies on the effective refractive index of the PSi multilayer produced by the contrast of the low/high porosity layers. Capture of a molecule within PSi increases the refractive index, causing a red shift in the spectral interference peaks. Therefore, our adhesion tests involved incubating the PSi samples with the peptides listed in table 1, all at 20 µM concentration, then measuring the optical reflectivity and quantifying the red shift of the cavity resonance. The efficiency of the peptide adsorption on PSi was assessed via the capture event of streptavidin by the biotin moiety displayed at the C-terminus of the peptides. Achievable sensitivity and minimum LOD to detect streptavidin is 10 nM.13 Figure 1 shows a typical reflectance spectra recorded at different steps of surface modification, after binding for example of the biotinylated PC4 peptide (SLVSHMQTGGGSK-Biotin) and subsequent streptavidin capture.


11

Langmuir

PSi PSi-HCl [..PC4-Biotin] [..Streptavidin-FITC]

1.2

1.0

Reflectance (a.u)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 25

0.8

0.6

0.4

0.2

0.0 800

900

1000

Wavelength (nm)

Figure 1. Reflectance spectra of porous silicon microcavities at different steps of its functionalization with peptide and capturing streptavidin. Molecule adsorption causes a red shift in the cavity resonance. Here the biotinilated PC4 peptide (SLVSHMQTGGGSK(Biotin)) was adsorbed onto PSi and used to bind streptavidin.

The first surface treatment step (PSi-HCl) resulted in a negative shift of about 5 nm in the reflectance spectrum due to opening of pores and removal of native oxide. When the biotinylated peptide bound to the PSi surface, the spectra shifted towards higher wavelengths (+6 nm) and the same effect was observed after streptavidin was bound to biotin. Table 1 gathers the measured shifts in the reflectance spectra of the PSi substrates after peptide binding (∆λPeptide) and capture of streptavidin (∆λStrep). Surprisingly, we obtained a negative shift after streptavidin incubation of microcavities treated with peptides truncated at their C-terminus, except for the longer peptide PN5, which still contained the glutamine residue (Q) in its sequence. We can interpret


12

Page 13 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

this negative shift as the consequence of the rinsing step that removes BSA molecules from the surface that are co-incubated with streptavidin. This result suggests either that peptides PN1-4 are not binding to PSi or that the configuration of the peptides does not expose correctly the biotin, impairing the capture of streptavidin. Contrariwise, positive shifts were measured for peptides PC1-7, analogues of the lead peptide P truncated by their N-terminus. The largest spectral shifts were recorded for the 12mer peptide P and the shorter analogue PC4 (SLVSHMQ T). Extraction of precise information on the quantity of bound molecules from these data is not trivial due to the highly variable sequence of the adsorbed peptides. To access some quantitative information, we performed fluorescence-based experiments: peptide modified PSi microcavities were used to capture the FITC tagged streptavidin and the fluorescence intensity was calculated from the corresponding images, presented in Fig. 2.


13

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 25

Figure 2. Adhesion of fluorescent streptavidin on the biotinylated peptides adsorbed on PSi samples, observed by ﬂuorescence microscopy. Scale bar 100 µm.


14

Page 15 of 25

2000

Fluorescence Intensity (a.u)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1500

1000

500

0 P

PC1 PC2 PC3 PC4 PC5 PC6 PC7 PN1 PN2 PN3 PN4 PN5

Peptides

Figure 3. Fluorescence intensities recorded for PSi microcavities functionalized with different biotinylated peptide derivatives after binding of fluorescent streptavidin. Peptide binding efficiencies measured via the fluorescence intensity corresponding to the N-terminus truncated form of P lead peptides (PC1-7), whereas those corresponding to the C-terminus truncated form (PN1-5) (only the variable peptide sequence is presented).

Fig 2 displays different fluorescent intensity images of the captured streptavidin onto PSi microcavities depending on the peptide analogue used for PSi functionalization. Given that the concentration of the streptavidin solution was the same in all the experiments, we can hypothesize that, the observed differences in the fluorescence intensity can be explained by the


15

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 25

amount of peptide adsorbing to the surface. The recovered fluorescence intensity was quantified by subtracting spectra of unspecific streptavidin binding from the signal recorded for each peptide- functionalized surface. The results are presented in Figure 3 and can be associated with the binding efficiency of various peptide derivatives onto the PSi microcavities. Both one way ANOVA and Homogeneity of Variance Test indicates p-value that is smaller than 0.05, hence each peptide group have significantly different means.

The highest fluorescence value was obtained with the octapeptide PC4 (SLVSHMQT), which is in agreement with the observed reflectance shifts (Table 1). PC4 is the original P peptide where the first four amino acids (SPGL) were subtracted. Peptide analogues truncated at the Cterminus and lacking Q and T residues (PN1-4) induced a significantly lower fluorescence. Both amino acids, Q, T are polar in nature that matches with the hydrophilic behavior of silicon. The adhesion behavior of the peptide is mainly defined by polar and other non-covalent interactions of individual residues with the surface; however peptide conformation also plays a role. Previous work showed that conformation of peptide changes when exchanging threonine with proline at the C terminus of the peptide, which significantly modifies its binding affinity towards Si(100).25 We cannot explicitly say that the peptide affinity is due to glutamine and threonine residues, but we presume that these two amino acids have a main contribution to the binding affinity. On the contrary, N-terminus truncations have less impact (PC1-7) on the fluorescence, revealing that it is probably the C-terminus part of the lead peptide P that is mostly involved in PSi binding. Though to a smaller extent, the N-terminus truncated peptide derivatives reveal variable fluorescence signals too. Subtracting serine S from the P peptide, leading to PC1, nearly halves the intensity and the absence of the next two amino acids P and G (PC2-3) decreases even more


16

Page 17 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

the fluorescence, pointing out the crucial role of these first three residues in peptide’s adhesion. However, when the following L is removed from the sequence, leading to the PC4 analogue, a very good signal is recovered. This might suggest that the remaining SLVSHMQT sequence can reveal a better configuration for enhanced adsorption.

Screening key amino acids involved in peptide affinity towards PSi The importance of the C-terminus and the key role of several amino acids in peptides’ binding towards n+ type Si were already predicted in our previous theoretical study.26 Molecular dynamic calculations indicated that any modification in the QT part destroys peptide affinity, whereas methionine (M) substitution in certain positions within the sequence may decrease or enhance affinity. Our present measurements have demonstrated the relevance of the C-terminus and the QT amino acids in the peptides’ binding efficiency (see poor adhesion of PN1-4 analogues in Fig. 3) though in this case against p+ type Si. This might indicate that doping of Si does not influence the strong interactions of the QT residues and C terminus with this material.

Our theoretical calculations and simulated residual scanning analysis have shown also that substitution of methionine and glutamine residues with both valine and alanine decreased the binding affinity of a peptide towards n+-type Si.25 To evaluate the role of these amino acids we substituted methionine and glutamine in the sequence of the best binder analogue PC4 with alanine and valine, respectively: peptides SLVSHMVT (PH1) and SLVSHAQT (PH2) were prepared (Table2) and biotinylated for further use. Adsorption of these peptides towards p+-Si was first monitored by the reflectance spectra of the PSi microcavities. The measured shifts


17

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 25

corresponding for the three PC4, PH1, and PH2 peptides are gathered in Table 2. As an example, the reflectance spectra after PH1 binding and streptavidin capture are shown in Fig 4 (a). No red shift was detected, indicating that PH1 was not binding to the surface. Unlike with PH1, red shifts were recorded for PH2, albeit with a lower value than those measured for PC4. This outcome is also confirmed by the fluorescence results, as depicted in Fig 4 (b).

Table 2. Truncated peptide sequences synthesized (substitution of M and Q residues in PC4) to screen key amino acids involved in adhesion towards PSi. The measured cavity resonance shifts of the PSi samples after adsorption of the biotinylated peptides and capture of streptavidin are also presented.

Id

Peptide

∆λPeptide (nm)

∆λStrep(nm)

PC4

SLVSHMQT

8.83 ± 0.29

14.6 ± 1.37

PH1

SLVSHMVT

-0.88 ± 0.09

1.81 ± 0.85

PH2

SLVSHAQT

3.12 ± 0.16

3.91 ± 1.48


18

Page 19 of 25

1 PSi PSi-HCl [..PH1-Biotin]

0.9

a)

b) 1400

Fluorescence Intensity (a.u)

0.7 0.6 0.5 0.4 0.3 0.2

1200 1000

0.1 0

SLVSHMQT (PC4) SLVSHMVT (PH1) SLVSHAQT (PH2)

[..Streptavidin-FITC]

0.8

Reflectivity (a.u)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

800 600 400 200 0 500

800

850

900 Wavelength (nm)

950

1000

600

Wavelength (nm)

Figure 4. Effects of Q and M substitution by V an A in PC4 sequence on binding efficiency towards PSi: a) reflectance spectra of PSi functionalized with the biotinylated PH1 (SLVSHMVTGGGSK(Biotin)-) – no or very weak adsorption b) Fluorescence spectra recorded after fluorescent streptavidin capturing on PSi treated with PC4 (black), PH1 (red) or PH2 (blue).

Figure 5. Capture of fluorescent streptavidine by the biotinylated peptides adsorbed on PSi observed by ﬂuorescence microscopy. Scale bar 100 µm.

The fluorescence is almost nonexistent when a PSi modified with PH1 (SLVSHMVT) peptide was used to capture FITC-labeled streptavidin, while the best results were obtained with the unmodified PC4 peptide. Substituting M with A in PH2 decreased the fluorescence by about 80 % in comparison to the result obtained using PC4 (Figs. 4b, 5). These results clearly indicate that


19

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 25

glutamine is crucial for binding and methionine is also contributing to the peptide affinity for PSi.

CONCLUSIONS The aim of our work was to identify and engineer the shortest peptide sequence revealing the highest affinity towards p+-Si for use as an efficient linker for biomolecules immobilization on Si. We examined the adhesion properties of fourteen different variants of the peptide SPGLSLVSHMQT that was previously identified by phage display as a high affinity binding peptide for p+-Si. C and N truncated peptide sequences, as well as two mutants displaying valine and alanine residues replacing glutamine and methionine were prepared. The peptides were modified at their C-termini with a spacer and a biotin moiety to capture FITC labeled streptavidin and enable thereby monitoring of peptides adhesion by fluorescence measurements. We demonstrated that the shortest sequence SLVSHMQT was able to bind more efficiently onto p+-Si than the original 12mer P peptide. Moreover, our results reveal the key role of the Cterminus, the glutamine and threonine amino acids in the peptides’ binding to the p+-Si substrate. Results obtained with alanine-mutated peptides demonstrate that methionine is also a large contributor in the process of the peptide’s affinity for p+-Si. This work validates our previous molecular dynamic simulations where the importance of these residues in peptides’ adhesion against Si has been already hypothesized. In summary, we demonstrated that identifying the key amino acids responsible for peptides affinity against Si enables engineering of short peptides with improved adhesion compared to those elaborated via phage display technology. Use of such efficient linkers with enhanced recognition properties for a target material might find wide applications in their controlled


20

Page 21 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

interfacing with biomolecules. The presented methodology can be extended for various materials and combined with molecular dynamic calculations presents a powerful tool for the design of novel, short peptide sequences with predictable functionalities towards a given target. Our work paves the way for the use of peptides in numerous material-engineering applications, such as predictable nanocrystals growth, elaboration of functional biosensors with enhanced sensitivity and efficient drug delivery systems.

ASSOCIATED CONTENT Supporting Information. Supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Csilla Gergely *To whom correspondence should be addressed. [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


21

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 25

ACKNOWLEDGMENTS We would like to thank Jade Francois for her help in peptide synthesis during her internship. We are thankful for the support of computational resources provided by Centre Informatique National de l’Enseignement Supérieur (CINES). ABBREVIATIONS TLC, thin layer chromatography; DMF, Dimethylformamide, Fmoc SPPS , 9fluorenylmethyloxycarbonyl Solid Phase Peptide Synthesis; DIEA, Diisopropylethylamine; DMSO, Dimethylsulfoxyde; BOP, (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; DCM , Dichloromethane.

REFERENCES (1)

Lin, A. Y.-M.; Chen, P.-Y.; Meyers, M. A. The Growth of Nacre in the Abalone Shell. Acta biomaterialia 2008, 4, 131–138.

(2)

Busch, S. Regeneration of Human Tooth Enamel. Angewandte Chemie International Edition 2004, 43, 1428–1431.

(3)

Palmer, L. C.; Newcomb, C. J.; Kaltz, S. R.; Spoerke, E. D.; Stupp, S. I. Biomimetic Systems for Hydroxyapatite Mineralization Inspired by Bone and Enamel. Chemical Reviews 2008, 108, 4754–4783.

(4)

Dickerson, M. B.; Sandhage, K. H.; Naik, R. R. Protein-and Peptide-Directed Syntheses of Inorganic Materials. Chemical reviews 2008, 108, 4935–4978.

(5)

Hollister, S. J. Porous Scaffold Design for Tissue Engineering. Nature materials 2005, 4, 518–524.

(6)

Luz, G. M.; Mano, J. F. Biomimetic Design of Materials and Biomaterials Inspired by the Structure of Nacre. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 2009, 367, 1587–1605.

(7)

Sewell, S. L.; Wright, D. W. Biomimetic Synthesis of Titanium Dioxide Utilizing the R5 Peptide Derived from Cylindrotheca F Usiformis. Chemistry of materials 2006, 18, 3108–


22

Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

3113. (8)

Krӧger, N.; Deutzmann, R.; Sumper, M. Polycationic Peptides from Diatom Biosilica That Direct Silica Nanosphere Formation. Science 1999, 286, 1129–1132.

(9)

Inoue, H.; Ozaki, N.; Nagasawa, H. Purification and Structural Determination of a Phosphorylated Peptide with Anti-Calcification and Chitin-Binding Activities in the Exoskeleton of the Crayfish, Procambarus Clarkii. Bioscience, biotechnology, and biochemistry 2001, 65, 1840–1848.

(10)

Reiss, B. D.; Mao, C.; Solis, D. J.; Ryan, K. S.; Thomson, T.; Belcher, A. M. Biological Routes to Metal Alloy Ferromagnetic Nanostructures. Nano letters 2004, 4, 1127–1132.

(11)

Chen, C.-L.; Zhang, P.; Rosi, N. L. A New Peptide-Based Method for the Design and Synthesis of Nanoparticle Superstructures: Construction of Highly Ordered Gold Nanoparticle Double Helices. Journal of the American Chemical Society 2008, 130, 13555–13557.

(12)

Behrens, S. S. Synthesis of Inorganic Nanomaterials Mediated by Protein Assemblies. Journal of Materials Chemistry 2008, 18, 3788–3798.

(13)

Estephan, E.; Saab, M.-B.; Agarwal, V.; Cuisinier, F. J.; Larroque, C.; Gergely, C. Peptides for the Biofunctionalization of Silicon for Use in Optical Sensing with Porous Silicon Microcavities. Advanced Functional Materials 2011, 21, 2003–2011.

(14)

Naik, R. R.; Stringer, S. J.; Agarwal, G.; Jones, S. E.; Stone, M. O. Biomimetic Synthesis and Patterning of Silver Nanoparticles. Nature materials 2002, 1, 169–172.

(15)

Chiu, C.-Y.; Li, Y.; Ruan, L.; Ye, X.; Murray, C. B.; Huang, Y. Platinum Nanocrystals Selectively Shaped Using Facet-Specific Peptide Sequences. Nature chemistry 2011, 3, 393–399.

(16)

Tomczak, M.; Slocik, J.; Stone, M.; Naik, R. Bio-Based Approaches to Inorganic Material Synthesis. Biochemical Society Transactions 2007, 35, 512–515.

(17)

Willett, R.; Baldwin, K.; West, K.; Pfeiffer, L. Differential Adhesion of Amino Acids to Inorganic Surfaces. Proceedings of the National Academy of Sciences 2005, 102, 7817– 7822.

(18)

Heinz, H.; Farmer, B. L.; Pandey, R. B.; Slocik, J. M.; Patnaik, S. S.; Pachter, R.; Naik, R. R. Nature of Molecular Interactions of Peptides with Gold, Palladium, and Pd- Au Bimetal Surfaces in Aqueous Solution. Journal of the American Chemical Society 2009, 131, 9704–9714.

(19)

Ramakrishnan, S. K.; Martin, M.; Cloitre, T.; Firlej, L.; Cuisinier, F. J.; Gergely, C. Insights on the Facet Specific Adsorption of Amino Acids and Peptides toward Platinum.


23

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 25

Journal of chemical information and modeling 2013, 53, 3273–3279. (20)

Oren, E. E.; Tamerler, C.; Sahin, D.; Hnilova, M.; Seker, U. O. S.; Sarikaya, M.; Samudrala, R. A Novel Knowledge-Based Approach to Design Inorganic-Binding Peptides. Bioinformatics 2007, 23, 2816–2822.

(21)

Ruan, L.; Ramezani-Dakhel, H.; Chiu, C.-Y.; Zhu, E.; Li, Y.; Heinz, H.; Huang, Y. Tailoring Molecular Specificity toward a Crystal Facet: A Lesson from Biorecognition toward Pt 111. Nano letters 2013, 13, 840–846.

(22)

Seker, U. O. S.; Wilson, B.; Dincer, S.; Kim, I. W.; Oren, E. E.; Evans, J. S.; Tamerler, C.; Sarikaya, M. Adsorption Behavior of Linear and Cyclic Genetically Engineered Platinum Binding Peptides. Langmuir 2007, 23, 7895–7900.

(23)

Pellegrini, V.; Tredicucci, A.; Mazzoleni, C.; Pavesi, L. Enhanced Optical Properties in Porous Silicon Microcavities. Physical Review B 1995, 52, R14328.

(24)

Amblard, M.; Fehrentz, J.-A.; Martinez, J.; Subra, G. Methods and Protocols of Modern Solid Phase Peptide Synthesis. Mol. Biotechnol. 2006, 33, 239–254.

(25)

Bachmann, M.; Goede, K.; Beck-Sickinger, A. G.; Grundmann, M.; Irbäck, A.; Janke, W. Microscopic Mechanism of Specific Peptide Adhesion to Semiconductor Substrates. Angew. Chem. Int. Ed. Engl. 2010, 49, 9530–9533.

(26)

Ramakrishnan, S. K.; Martin, M.; Cloitre, T.; Firlej, L.; Gergely, C. Molecular Mechanism of Selective Binding of Peptides to Silicon Surface. Journal of chemical information and modeling 2014, 54, 2117–2126.


24

Page 25 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

SYNOPSIS.


25

Engineered Adhesion Peptides for Improved Silicon Adsorption

Recommend Documents