Fluorimetric Studies of a Transmembrane Protein and Its Interactions

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Fluorimetric Studies of a Trans-Membrane Protein and Its Interactions with Differently Functionalized Silver Nanoparticles Marta Gambucci, Luigi Tarpani, Giulia Zampini, Giuseppina Massaro, Morena Nocchetti, Paola Sassi, and Loredana Latterini J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b02599 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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The Journal of Physical Chemistry

Fluorimetric Studies of a Trans-Membrane Protein and its Interactions with Differently Functionalized Silver Nanoparticles Marta Gambuccia, Luigi Tarpania, Giulia Zampinia, Giuseppina Massaroa,§, Morena Nocchettib, Paola Sassia, Loredana Latterini*a a

Dipartimento di Chimica, Biologia e Biotecnologie, Università di Perugia - Via Elce di Sotto, 8,

06123 Perugia, Italy b

Dipartimento di Scienze Farmaceutiche, Università di Perugia, Via del Liceo 1, 06123 Perugia,

Italy. §

Present address: Henkel ICIQ Joint Unit - ICIQ Institute of Chemical Research of Catalonia, Tarragona, Spain

* Corresponding author. Phone: +39-75-5855214; Fax: +39-75-5855598. E-mail: [email protected] (L. Latterini)

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Abstract Trans-membrane proteins play important roles in the inter-cellular signaling to regulate the interactions among adjacent cells and influence cell fate. The study of the interactions between membrane proteins and nanomaterials is paramount for the design of nanomaterial-based therapies. In the present work, the fluorescence properties of the trans-membrane receptor Notch2 have been investigated. In particular, steady state and time resolved fluorescence methods have been used to characterize the emission of tryptophan residues of Notch2 and then this emission is used to monitor the impact of silver colloids on protein behavior. To this aim, silver colloids are prepared with two different methods to make sure they bear hydrophilic (citrate ions, C-AgNPs) or hydrophobic (dodecanethiol molecules D-AgNPs) capping agents; the preparation procedures are tightly controlled in order to obtain metal cores with similar size distributions (7.4 ± 2.5 and 5.0 ± 0.8 nm, respectively), thus making easier the comparison of the results. The occurrence of strong interactions between Notch2 and D-AgNPs is suggested by the efficient and statistically relevant quenching of the stationary protein emission already at low nanoparticle concentrations (ca. 12% quenching with [D-AgNPs] = 0.6nM). The quenching becomes even more pronounced (ca. 60%) when [D-AgNPs] is raised to 8.72nM. On the other hand, the addition of increasing concentrations of C-AgNPs to Notch2 does not affect the protein fluorescence (intensity variations below 5%) indicating that negligible interactions are taking place. The fluorescence data, recorded in the presence of increasing concentrations of silver nanoparticles, are then analyzed through the Stern-Volmer equation and the sphere of action model to discuss the nature of the interactions. The effect of D-AgNPs on the fluorescence decay times of Notch2 is also investigated and a decrease of the average decay time is observed (from 4.64 to 3.42 ns). The observed variations of the stationary and time-resolved fluorescence behavior of the protein are discussed in terms of static and collisional interactions. These results document that the capping shell is able to drive the protein-particle interactions, which have likely a hydrophobic nature.


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Introduction Proliferation and differentiation are fundamental processes in cell life cycle, thus aberrations in any biological pathway regulating one of these processes may lead to uncontrolled cell growth and consequent development of diseases. On the other end, a meticulously planned regulation of these pathways is essential to the development of new tissue regeneration methods. One of the mechanisms through which cell proliferation and differentiation are regulated depends on conformational changes in transmembrane receptors, therefore a deeper understanding of the effect of ligands and other potential therapeutic agents (such as nanoparticles) could lead to a breakthrough in both therapy and tissue engineering fields. Notch receptors are an important class of transmembrane proteins with large extracellular domains, which are involved in inter-cellular signaling to regulate cell proliferation and differentiation1,2. Notch signaling pathway and its dysfunction is thought to be partially responsible for the genesis and growth of many different types of tumor2,3. The intracellular domain of Notch is a membrane-bound transcription factor: ligand binding promotes two proteolytic cleavage events in the Notch receptor, resulting in the release of the intracellular domain2. A family of metalloproteases catalyzes the first cleavage. The outcome of genetic studies suggests that metal complexes bind to Notch and induce important conformational changes able to interfere with cell functioning4. Furthermore, the presence of multiple tryptophan residues in Notch2 receptor enables the detection of conformational changes using non-invasive and highly sensitive fluorescence techniques5,6. The chances to explore how nanomaterials interact with membrane proteins can foster the engineering of colloids to actively mediate the cell communication processes. Plasmonic metal nanoparticles have been widely studied and used for biomedical applications7-9; among the different metal colloids, silver nanoparticles have demonstrated particularly interesting biomedical and optical properties10-12. The use of these nanomaterials as tools for diagnostic and therapeutic purposes implies that they will are able to interact with biological substrates, such as lipid membranes, proteins or nucleic acids, whose structure and function might be affected. Indeed, recent studies13,14 have demonstrated that silver nanoparticles affect cell morphology, genetic material and biological processes at concentrations as low as 1-25 mg/L, even if concentrations of AgNPs higher than 25-50 mg/L are required to diminish significantly cell viability15,16. However, limited molecular information is available to exploit the parameters that control the interactions. While significant work has been done in order to understand the effects of these interactions on the properties of the nanoparticles17-21, little is known21 about the consequences of these interactions on protein properties and behavior, especially with regard to membrane proteins, like Notch2. This



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aspect deserves a deep understanding since protein folding can result in their reduced functionality or leads to the development of wrong biological signals. Literature presents various examples of the use of these techniques for the study of proteinnanoparticles interaction6,19,22-25, focusing mostly on serum proteins, such as Bovine Serum Albumin (BSA), often chosen as model proteins for the study of nanoparticles-proteins interaction, due to their availability, their binding capacities23,26-28. As stated in these examples, the interactions of BSA with metal nanoparticles (specifically silver) generally lead to fluorescence quenching and to a blue shift of the fluorescence maximum. The quenching mechanism can be quite complex and the formation of protein-particle adsorption complexes leading to particle aggregation has been proposed19,25,29. It has been reported that BSA unfolding occurs on metal nanoparticle surface and it fosters nanoparticles aggregation19,30. In this context, the surface chemistry of the metal colloids has been demonstrated to play a paramount role on driving the interactions between nanomaterials and proteins31,32. This work aims at investigating the interactions between a membrane protein, namely Notch2, and silver nanoparticles (AgNPs) by monitoring the fluorescence properties of the protein. The role of surface chemical properties of the colloids has been evaluated using AgNPs with different capping agents; in particular, AgNPs are prepared with two chemical procedures to obtain colloids capped with hydrophobic or hydrophilic (dodecanethiol or citrate ions) agents. The effects of hydrophobic and hydrophilic AgNPs on the fluorescent behavior of Notch2 are analyzed and discussed in terms of protein conformation changes.

Experimental section Materials Silver nitrate (99% purity), 1-dodecanethiol (98% purity), sodium borohydride (98% purity) and Notch2/chimera from rat (90% purity) were purchased from Sigma Aldrich. Trisodium citrate (99% purity) was obtained from Fluka. All aqueous solutions were prepared with Milli-Q water.

Synthesis of silver nanoparticles Silver nanoparticles (AgNPs) were prepared through a chemical reduction method. Hydrophilic AgNPs were synthesized by modifying the procedure reported by Natan et al. for gold nanoparticles33. 0.72 mL of a NaBH4 aqueous solution (0.1 M) was quickly added to 20 mL aqueous solution containing AgNO3 (2.5x10-3 M) and citrate (2.5x10-3 M) under vigorous stirring. The sudden color change of the solution to yellow indicates the formation of AgNPs. 4 ACS Paragon Plus Environment

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Hydrophobic AgNPs were prepared at low temperature following the strategy proposed by Kang and coworkers34, with minor changes. In a typical procedure, the reaction was carried out at 0°C, by mixing 10 mL of an AgNO3 solution in ethanol (3x10-2 M) and 0.05 mL of dodecanethiol under continuous stirring. Then a saturated solution of NaBH4 in ethanol (previously cooled at 0°C) was slowly added to the solution. The reaction mixture was stirred for 2h maintaining the temperature at 0°C and then stored -18°C for 4h; afterwards the nanoparticles were precipitated by centrifugation and washed with ethanol.

Protein sample preparation Notch2 stock solution (0.1 mg/mL) was prepared in phosphate buffered saline (PBS, pH 7.4) and diluted to 0.003 mg/mL before use.

Instrumentation A Philips transmission electron microscope (mod. 208, operating at 80 kV of beam acceleration) is used to analyze the nanoparticle size distribution. The nanoparticles suspensions are deposited on a 400-mesh copper coated with formvar support grid and are left overnight in a desiccator to allow the solvent evaporation. The size distribution histograms of the samples are obtained by analyzing at least 150-200 nanoparticles for each colloid. The dispersion of particle dimensions has been evaluated by the standard deviation (σ) of the fitted Gaussian distribution. An atomic force microscope (Solver-Pro P47H, NT-MDT) is used to record topographic images of the prepared nanoparticles. Samples for AFM analysis are prepared placing a drop of the NPs suspension on a mica surface and spin-coated in order to spread the samples during solvent removal. The measurements are carried out in semicontact conditions by use of a 190-325 kHz cantilevers. The size distribution histograms of the samples are obtained by analyzing at least 100150 nanoparticles for each colloidal sample. Quantitative determination of Ag is performed with a Varian 700-ES series inductively coupled plasma-optical emission spectrometer (ICP-OES). A weighed amount of AgNP is dissolved in concentrated HNO3 and the solution is diluted to a final volume of 100 mL before ICP analysis. Absorption spectra of nanoparticles are recorded using a Perkin-Elmer Lambda 800 double beam spectrophotometer. Fluorescence excitation and emission spectra of the protein in the presence of increasing concentrations of silver colloids are obtained with a Horiba Fluoromax fluorimeter, using a rightangle configuration. All measurements are taken using a 1 cm path length quartz cuvette. To properly investigate and compare the effect of AgNPs on the photophysical behavior of the 5 ACS Paragon Plus Environment


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proteins, excitation and emission spectra of the protein are corrected for the dilution and the fraction of absorbed light. Fluorescence decay times of the protein are measured through Single Photon Counting using an Edinburgh Instruments 199 spectrophotometer with a 295 nm LED as pulsed light source. Notch2 emission was observed at 335 nm and at 350 nm. A 290 nm cut-off filter was used to prevent the interference of the excitation light. The results are analyzed through multi-exponential fluorescence decay fitting using nonlinear least-squares error minimization analysis. The quality of the fit is assessed by the chi-squared values and the distribution of the residuals.

Results and Discussion Notch2 fluorescence properties The fluorescence behavior of aromatic amino acid residues is a well-established tool to investigate protein conformational changes5. The transmembrane protein Notch2 contains 21 Trp residues and 57 Tyr residues, so fluorescence can be used to investigate the protein behavior, but limited information is available on the fluorescence properties of this protein. Figure 1 shows the emission spectra of Notch2 in PBS buffer at different excitation wavelengths; Notch2 presents a quite broad emission band, centered at 335 nm when excited at 280 nm. Upon excitation the protein at 295 nm (Figure 1), the fluorescence spectrum has the maximum at 342 nm. This bathochromic shift is attributed to the lack of contribution from Tyr residues in the latter spectrum, since absorption of Tyr is negligible at 295 nm; the contribution of tyrosinate species, eventually formed upon proton transfer, cannot be excluded. However, the integrated intensity of tyrosinate has a limited contribution due to its short decay times5 (see Figure S1). The Notch2 spectrum, recorded upon excitation at 295 nm, presents a quite broad shape and a full width at half maximum (FWHM) value of 6030 cm-1 is measured, which is about 20% wider than that of Trp in water5. This broadening is likely related to the presence of multiple Trp contributions in the fluorescence spectrum of Notch2, suggesting that the 21 Trp residues have slightly different micro-environments; the position of the spectrum maximum suggests that the intrinsic fluorescence of the Notch2 is mainly determined by the emission of Trp residues exposed to a more polar environment. Fluorescence decay measurements on Notch2 are also performed. Despite the high number of Trp residues, the fluorescence decay of Notch2, recorded in the present experimental conditions, is satisfactorily reproduced by bi-exponential functions, although the most correct method to analyze the data would be in terms of decay time distributions. The bimodal emission decay model likely 6 ACS Paragon Plus Environment

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represents the average decay times of the residues with higher fluorescence efficiencies in the used experimental conditions35. The obtained decay times are shown in Table 1. As reported for other proteins,5 Notch2 decay times increase at longer emission wavelengths, where a major contribution of Trp residues exposed to the polar solvent can be expected. This observation indicates that the buried Trp residues in Notch2 are quenched likely due to the interactions of with the surrounding environment (peptide bonds and amino-acid side chains are known to quench Trp fluorescence5).

1,0

0,8

Intensity (arb.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


0,6

0,4

0,2

0,0 300

350

400

450

Wavelength (nm)

Figure 1: Emission spectra of Notch2 in aqueous solution (pH 7.4): λexc= 280 nm (black line, open squares) and λexc= 295 nm (red line, open circles).

Table 1: Fluorescence decay times of Notch2 at different emission wavelength (λexc= 295 nm). λem (nm)

τF (ns)

335

5.71 ± 0.08 (61%) 1.08 ± 0.04 (39%) 1.0197

3.90

350

6.81 ± 0.06 (60%) 1.39 ± 0.04 (40%) 1.0351

4.64

ChiSQ

(ns)

Synthesis and Characterization of silver nanoparticles Silver nanoparticles (AgNPs) are synthesized following two different procedures in order to obtain colloids bearing capping agents able to impart hydrophilic or hydrophobic properties to the colloids (see the experimental section). Thus, AgNPs are synthesized in water using citrate anion as capping agents and in ethanol taking advantage of the stabilizing capacities of dodecanethiol. The synthetic conditions have been carefully optimized to achieve samples with similar morphological properties. 7 ACS Paragon Plus Environment


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The prepared nanoparticles have been characterized by TEM and AFM measurements to accurately determine their dimensional properties. The concentrations of the colloids in each sample have been estimated through inductively coupled plasma (ICP) analysis of the suspensions and taking advantage of the average size data. TEM images of the citrate-capped AgNPs (C-AgNPs) show that the sample is constituted by spherical nanoparticles (Figure 2a). From TEM images, the size distribution of the metal core is obtained (Figure 2b, red bars), which is analyzed through Gaussian functions; this analysis evidences the presence of a single particle population and provides an average size of 7.4 nm with a standard deviation (σ) value of 2.5 nm. Analysis of the AFM images recorded on C-AgNPs gives an average height of 12.7 nm (σ = 9.6) for the colloidal system, which accounts for the size of the metal core and the capping shell (Figure 2b, grey bars). The comparison of the average dimensions, determined by TEM and AFM investigations, suggests that citrate molecules form a capping shell with an average thickness of about 2.7 nm. Since this value is bigger that the size of single citrate ion lying flat on the surface (~ 0.72 nm)36, the formation of multiple ionic layers around the particles cannot be excluded. In the obtained suspension, an elemental silver amount of 1.019×10-6 M was measured; this value, together with the average dimensions of the metallic core (obtained by analyzing the TEM data), allows to determine the concentration of C-AgNPs, which turns out to be 1.2×10-8 M (see Supporting Information for details).

Figure 2: (a) TEM image of C-AgNPs; (b) size distribution of C-AgNPs obtained from TEM images (red bars) and from AFM images (grey bars).


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TEM images of dodecanethiol-stabilized AgNPs (D-AgNPs), presented in Figure 3a, show highly monodispersed spherical nanoparticles; the diameter distribution (Figure 3b, red bars) was reproduced with a Gaussian function centered at 5.0 nm (σ = 0.8 nm). Furthermore, an average size of 9.1 nm (σ = 3.8 nm) for the colloidal system was obtained from AFM analysis (Figure 3b, grey bars). Since the capping shell has an estimated length of about 2.0 nm, the formation of multilayer coat around the metal nuclei cannot be excluded, also in this case. Through ICP measurements, the Ag content of the suspension is measured to be 2.396×10-6 M, from which a nanoparticles concentration value of 1.2×10-8 M is determined for D-AgNPs (see Supporting Information).

Figure 3: (a) TEM image of D-AgNPs; (b) Size distribution of D-AgNPs obtained from TEM images (red bars) and from AFM images (grey bars).

Table 2 summarizes the dimensional properties of the two AgNPs samples. It has to be noted that despite the different chemical nature of the capping agents, the control of the synthetic procedure enabled the achievement of silver colloids with the same morphology and similar average dimensions, although C-AgNPs present a broader size distribution of the metal nuclei.



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Table 2: Size and optical properties of C-AgNPs and D-AgNPs samples. Mean NPs sample

size

a

(nm)

σa (nm)

Average b

height (nm)

σb (nm)

Shell thickness (nm)

C-AgNPs

7.4

2.5

12.7

9.6

2.7

D-AgNPs

5.0

0.8

9.1

3.8

2

a

Determined through the analysis of TEM images

b

Determined through the analysis of AFM images

The silver colloids have been further characterized by extinction spectra (Figure S1a). The UVVisible spectrum of C-AgNPs in aqueous suspension presents an asymmetric band with the maximum at 388 nm, while the extinction spectrum of D-AgNPs dispersed in chloroform shows a single symmetric plasmonic band centered at 440 nm; the band symmetry is related to the size distribution of the two samples. The profile of the plasmonic band is extremely dependent on the homogeneous and stable dispersion of the colloids in the solvent, which is mainly controlled by the solvation properties of the capping agents. When D-AgNPs are injected in Milli-Q water, the metal colloids sediment rapidly; the colloidal dispersion of D-AgNPs in water is stable only in the presence of a small fraction of ethanol (5%) as documented by the extinction spectrum (Figure S1b), which presents the maximum at 445 nm.

Effects of AgNPs on the fluorescence properties of Notch2. The emission properties of Notch2 are investigated in PBS buffer (pH 7.4) suspensions, in the presence of increasing concentrations of C-AgNPs or D-AgNPs to evaluate the impact of silver colloids on structure of the protein (Figure 4). The addition of C-AgNPs has no significant effects on the fluorescence emission and excitation spectra of Notch2 (Figure 4a and Figure S3a). The minor changes, observed in the spectra when CAgNPs are added, are of the same magnitude as those observed for additions of sodium citrate (Figure S4); they are therefore not ascribable to the interactions of the protein with the metallic colloids. These data suggest that no interactions are taking place between C-AgNPs and the membrane protein, in the used experimental conditions.


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On the other hand, D-AgNPs have a larger effect on Notch2 fluorescence intensity, causing a quenching of the emission band even at small concentrations (Figure 4b). This conclusion is further supported by the lack of any effects on the Notch2 spectral behavior upon addition of neat dodecanethiol or ethanol (same concentrations used in the colloidal suspensions; Figure S5 and S6), leading to the conclusions that the changes of the fluorescence properties of the protein can certainly be attributed to the interactions with the colloidal D-AgNPs.

(a)

(b) 120000

Intensity (arb.u.)

120000

Intensity (arb.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


80000

40000

0

80000

40000

0 300

325

350

375

400

425

450

300

Wavelength (nm)

325

350

375

400

425

450

Wavelength (nm)

Figure 4: (a) Emission spectra (λexc= 280 nm) of Notch2 PBS buffer (pH 7.4) in the presence of increasing concentrations of C-AgNPs (0 to 0.57 nM); (b) emission spectra (λexc= 280 nm) of Notch2 in the presence of increasing concentrations of D-AgNPs (0 to 0.60 nM).

The Notch2 - D-AgNPs system was further characterized by expanding the range of concentrations of D-AgNPs and by measuring fluorescence decay times of Notch2 in the presence of the nanoparticles. Figure 5 shows the fluorescence spectra of the Notch2 in the presence of D-AgNPs up to 8.72 nM. The spectra show a remarkable intensity decrease and a narrowing of the spectral shape on the red side without an evident shift of the maximum (Figure 5; FWHH (Notch2) = 5564 cm-1; FWHH (Notch2+8.72 nM D-AgNP) = 5338 cm-1), thus indicating that the either the blue edge of the emission spectrum is enhanced or the contribution of the long wavelength emitters is more efficiently quenched. The support to the latter hypothesis is obtained from the analysis of time resolved fluorescence data.



(a)

(b) 1,0

Normalized intensity (arb.u.)

120000

Intensity (arb.u.)

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80000

40000

0

0,8

0,6

0,4

0,2

0,0 300

325

350

375

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425

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300

325

Wavelength (nm)

350

375

400

425

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Wavelength (nm)

Figure 5: (a) Emission spectra (λexc= 280 nm) of Notch2 PBS buffer (pH 7.4) in the presence of increasing concentrations of D-AgNPs (0 to 8.72 nM); (b) normalized emission spectra of Notch2 (black line, open squares) and Notch2 + 8.72 nM of D-AgNPs (orange line, open triangles).

Table 3 shows the decay parameters, obtained by adequately reproducing the fluorescence decays of Notch2, in the presence of increasing amounts of D-AgNPs through bi-exponential functions. The data highlight that the long component of the decay progressively decreases in the presence of DAgNPs. These data together with the fluorescence intensity quenching, observed in red-side of the emission spectra, confirm the efficient quenching of the more exposed Trp residues.

Table 3: Fluorescence decay parameters obtained by fitting Notch2 fluorescence decay curves recorded in PBS buffer (pH 7.4) in the presence of increasing concentrations of D-AgNPs (λexc= 295 nm, λem= 350 nm). D-AgNPs concentration

τF (ns)

ChiSQ

(ns)

(nM) --

6.81 ± 0.06 (60%) 1.39 ± 0.04 (40%) 1.0351

4.64

1.81

6.30 ± 0.06 (60%) 1.24 ± 0.04 (40%) 1.0515

4.28

4.47

6.1 ± 0.1 (56%)

1.28 ± 0.04 (44%) 1.0244

3.98

8.72

5.3 ± 0.1 (53%)

1.3 ± 0.1 (47%)

3.42

1.0039


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Quantitative analysis of Notch2-AgNPs interaction. The intermolecular quenching processes can be either dynamic and static in nature. Dynamic (or collisional) quenching is the deactivation of a fluorophore in the excited state upon contact or collision with a quencher in suspension. Static quenching is associated to the formation of a groundstate non-fluorescent complex between the fluorophore and quencher but also to long-range interactions, which lead to excitation quenching by circumventing diffusional processes. In the latter case, the quencher suppresses fluorescence within a finite action volume. In order to have deeper insights on the nature of the interactions between Notch2 and AgNPs, the fluorescence data have been analyzed through the Stern-Volmer equation (Eq. 2)

= 1 + = 1 +

(Eq. 2)

where I0 and I are the integrated fluorescence intensities in the absence or in the presence of increasing concentrations of AgNPs respectively, τ0 and τ are the average fluorescence decay times recorded in the absence or in the presence of increasing concentrations of AgNPs respectively, and KSV = kq × τ0, where kq is the bimolecular quenching constant). Figure 6a reports the Stern-Volmer plots of the fluorescence intensity for Notch2 in the presence of C-AgNPs and D-AgNPs. The plots show different quenching efficiencies, as documented by the KSV values (Table 4), which differ by two orders of magnitude. Instead, Figure 6b collects the Stern-Volmer plots obtained through steady-state and time-resolved fluorescence data of Notch2 upon addition of increasing concentrations of D-AgNPs; the plots evidence different quenching efficiencies, being steady-state KSV almost one order of magnitude higher than time-resolved KSV (Table 4). This distinct behavior in the quenching of the stationary and time-resolved fluorescence data is generally associated with the coexistence of different interaction mechanisms, such as static quenching and dynamic mechanism. The data have been further analyzed through an implemented Stern-Volmer relation, which accounts for dynamic and static interactions and enables to evaluate the sphere of action volume (Eq. 3); in particular, the parameter KD accounts for the dynamic contribution, while the molar volume V defines the volume within which the quencher must be located to quench the fluorescence of the proteins and corresponds to an active volume element surrounding the excited molecules. This second parameter represents the static quenching component of the model.



= 1 + ×

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(Eq. 3)

The fitting parameters are collected in Table 4. When the sphere of action model has been applied to the data recorded from the Notch2-C-AgNPs system, the primacy of dynamic interactions is confirmed by the large value of KD (comparable with static Ksv) and the small contribution of the molar volume (V ~ 9×101 M-1; see Table 4). On the other hand, the data indicate that the interactions between D-AgNPs and Notch2 are dominated by the static component, being the KD value negligible compared to V. Moreover, the molar volume obtained from the fit (V ~ 108 M-1) leads to a sphere of action of µm-dimension which is orders of magnitude larger than the nanoparticle size. This would be possible if aggregation of the nanoparticles occurred in the presence of protein, or protein molecules were adsorbed on the particles surface. The hypothesis of protein adsorption on the colloids is the most probable, in agreement with the recent findings at single molecule level19. However, differently from what observed in the cited work, we have no evidences that the adsorption process of the protein is activated by aggregation of nanoparticles (see Figure S2). The occurrence of relevant interactions between the protein and the colloids stabilized by hydrocarbon chains, agrees with the hypothesis that these interactions have a strong hydrophobic nature. In particular, the hydrophobic shell of the colloids might drive the interactions with the hydrophobic portion of the protein, thus leading the metal nuclei in close contact with the functional core of the protein.37 In conclusion, the presented results show that only hydrophobic silver colloids are able to interact with Notch2 protein and affect its behavior already at very low concentrations, which is well below the toxicity levels13-16; overall, these results highlight that the efficiency of the interactions between membrane protein and colloids is strongly modulated by the chemical nature of the stabilizing shell.


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[AgNP] (mg/L)

(a)

0,0

0,5

1,0

1,5

[D-AgNP] (mg/L)

(b)

2,0

1,20

0

5

0,0

2,0E-9

10

15

20

6,0E-9

8,0E-9

3,0 2,8

1,15

2,6 2,4

1,10

I0/I or τ0/τ

2,2

I0/I

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


1,05

1,00

2,0 1,8 1,6 1,4

0,95

1,2 1,0

0,90 0,0

2,0E-10

4,0E-10

6,0E-10

8,0E-10

1,0E-9

[AgNP] (M)

4,0E-9

[D-AgNP] (M)

Figure 6: (a) Stern-Volmer plots built using the integrated intensity values of Notch2 emission band with increasing concentrations of C-AgNPs (squares) and D-AgNPs (circles); (b) SternVolmer plots built using the integrated intensity values (solid symbols) and average decay times values (open symbols) for the addition of D-AgNPs to Notch2.

Table 4: Fitting parameters obtained by sphere of action model. System

KSVa

KSVb

KD (M-1) a

V (M-1) a

Notch2-C-AgNPs

1.2× 106

---

7.6× 105

9.0× 101

Notch2-D-AgNPs

1.7× 108

4.0× 107

1.1 × 102

1.1 × 108

a

Obtained from steady state data.

b

Obtained from time-resolved data.

Conclusions The fluorescence properties of Notch2, a transmembrane protein involved in intercellular signaling, have been investigated through steady state and time resolved techniques. The proteins, which includes many fluorescence amino acid residues, presents a quite broad spectrum with maxima around 340 nm and a bi-exponential fluorescence decay, indicating that its emission properties are dominated by the exposed Trp residues. Fluorescence methods are then used to investigate the interactions of Notch2 with silver nanoparticles; the investigations have been carried out using silver colloids designed to have similar size distributions (average diameter in the 5-7 nm range) but hydrophilic citrate ions (C-AgNPs) or hydrophobic dodecanethiol molecules (D-AgNPs) as stabilizing agents.



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The interactions of C-AgNPs or D-AgNPs with Notch2 protein leads to different results: the addition of C-AgNPs has limited effect on Notch2 fluorescence, whereas D-AgNPs strongly interact with tryptophan residues of Notch2 and induce a pronounced quenching in the emission band, together with a blue shift and band narrowing in the fluorescence spectra of the protein. The analysis of the data, using the collisional (Stern-Volmer) or mixed collisional and static models (sphere of action), revealed that the quenching processes in the presence of hydrophobic particles are mainly due to static interactions, which might assist protein aggregation. These results clearly indicate that the type of interactions between proteins and metal colloids can be modulated by the chemical nature of the stabilizing agents. In particular, the hypothesis that hydrophobic interactions are spontaneously established between the colloids and this important protein, might open new approaches in the design of nanomaterials for nanomedicine applications.

Supporting Information. ICP analysis and determination of AgNPs concentrations; extinction spectra of AgNPs; emission and excitation spectra of Notch2 in solution and in the presence of AgNPs and reference materials; fluorescence decay curves.

Acknowledgements Authors thank the Ministero per l’Università e la Ricerca Scientifica (Rome) and the University of Perugia (Fondo d’Ateneo per la ricerca di base 2014) for financial support.

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TOC Graphic


Fluorimetric Studies of a Transmembrane Protein and Its Interactions

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