Nanoscale Mechano–Electronic Behavior of a Metalloprotein as a

Article pubs.acs.org/Langmuir

Nanoscale Mechano−Electronic Behavior of a Metalloprotein as a Variable of Metal Content Tatini Rakshit, Siddhartha Banerjee, Sourav Mishra, and Rupa Mukhopadhyay* Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India S Supporting Information *

ABSTRACT: In this work, we have explored an approach to finding a correlation between the mechanical response of a metalloprotein against a range of applied force (by force curve analysis) and its electrical response under pressure stimulation (by current sensing atomic force spectroscopy) at the nanoscale. Ironstorage protein ferritin has been chosen as an experimental model system because it naturally contains a semiconducting iron core. This core consists of a large number of iron atoms and is therefore expected to exert a clear influence on the overall mechanical response of the protein structure. Four different ferritins (apoferritin, Fe(III)-ferritins containing ∼750 and ∼1400 iron atoms, and holoferritin containing ∼2600 iron atoms) were chosen in order to identify any relation between the mechano−electronic behavior of the ferritins and their metal content. We report the measurement of Young’s modulus values of the ferritin proteins as applicable in a nanoscale environment, for the first time, and show that these values are directly linked to the iron content of the individual ferritin type. The greater the iron content, the greater the Young’s modulus and in general the slower the rate of deformation against the application of force. When compressed, all the four ferritins exhibited increased electronic conductivity. A correlation between the iron content of the ferritins and the current values observed at certain bias voltages could be made at higher bias values (beyond 0.7 V), but no such discrimination among the four compressed ferritins could be made at the lower voltages. We propose that only at higher voltages can the iron atoms that reside deeper inside the core of the ferritins be accessed. The iron atoms that could be situated at the inner wall of the protein shell appear to make a general contribution to the electronic conductivity of the four ferritin systems.

■

nm and an internal diameter of 7 to 8 nm.8 Ferritin can gather excess free iron in the form of ferrihydrite phosphate in its core to prevent the harmful accumulation of iron in the body.9 The core of mammalian holoferritin can contain up to 4500 iron atoms in the form of ferrihydrite phosphate [(FeOOH)8(FeOPO3H2)].8 This stored iron is released from holoferritin by means of the reductive dissolution of the core to supply the iron required for different cellular processes. Ferritin is structurally robust and stays in its functional form in an aqueous environment having a pH of between 4.0 and 9.0 and well up to a temperature of 80 °C10 and is therefore quite suitable for on-surface studies under varied experimental conditions. Though the mechanical properties of the ferritin proteins have not been extensively studied, it was reported earlier that the presence of ferritin nanoparticles in poly(vinyl alcohol) (PVA) nanofibers showed an enhancement of the elastic modulus as compared to that of pure PVA nanofibers because of chemical interactions between ferritin and the PVA matrix.11 Bhattacharyya et al. showed that composite films containing ferritin-functionalized multiwalled carbon nanotubes

INTRODUCTION Investigations of the mechanical properties of proteins (e.g., elasticity, deformability, compressibility, etc.) are important for utilizing proteins in different biomaterial applications. So far, only a few reports on understanding the nanoscale mechanical properties of globular proteins have been made.1−3 Although a number of studies on force-induced solid-state electron transport in globular proteins have been reported4,5 and it has been largely observed that the application of compressive force increases the solid-state conductivity of proteins,6,7 little or no information on a correlation between the elasticity of a protein and the force-induced conductivity increase has been offered. In this context, it would be interesting to study a metalloprotein that contains a large metal core consisting of a few thousand metal atoms because the presence of a large number of metal atoms is expected to give rise to distinctively different results in comparison to the metal-free apoprotein. Ferritins, the iron-storage proteins, naturally contain a few thousand iron atoms inside the protein shell. They can therefore be considered to be an excellent experimental model system for such a study. Ferritin is an omnipresent protein because it can be found in all animals, plants, and bacteria. It has a unique ordered arrangement of 24 subunits that leads to the formation of a hollow sphere with an external diameter of approximately 12 © 2013 American Chemical Society

Received: July 3, 2013 Revised: September 11, 2013 Published: September 12, 2013 12511

dx.doi.org/10.1021/la402522m | Langmuir 2013, 29, 12511−12519

Langmuir

Article

be 0.546 N/m. The probe was cleaned in a UV-ozone cleaner (Bioforce, Nanosciences) before imaging. The amplitude set point was 85−90% of the free oscillation amplitude, which is 11−13 nm (in fluid). The scan speed was 1.0−2.0 lines/s. All images presented here are raw data except for minimum processing limited to first-order to third-order flattening. The height values were measured as the difference between the highest point of the cross-sectional diagram and the average height of the substrate surface. Dimensional analysis was performed on 50−60 molecules from different images. To acquire information on the thickness of the ferritin films generated on bare and modified gold surface, we performed scratching experiments (details in Supporting Information). The force curves, consisting of 4000 data points, were obtained by imposing a maximum applied force of 2.0 nN (for Young’s modulus measurement). Then the amount of applied force was increased in a controlled manner starting from 10 to 70 nN with an interval of 10 nN at each stage using PicoView 1.12.2 software (for deformation study). The force−distance (F−D) experiments were repeated for ∼500 molecules from different areas as well as from different samples. In all of the experiments, a 35−45% humidity level was maintained. The results presented here have been obtained after analyzing 400−500 force curves for each system. To obtain the indentation values, the photodiode sensitivity was calibrated by obtaining a force curve on hard bare substrates (a freshly annealed Au(111) surface and a freshly cleaved HOPG surface) as reference. The HOPG substrate was used to estimate the effective radius of the AFM tip, which was found to be 7 nm. When a spherical tip scans a HOPG surface containing a step (step height 0.34 nm) lying along the direction perpendicular to the scan, if contact between both is established at a distance W, then W = (2RH − H2)1/2, where R is the tip radius and H is the height of the step. The tip radius can be determined by using the equation R = (H2 + W2)/2H (detailed steps in ref 24). After each measurement, the tip was checked to ensure that no change in the tip integrity (i.e., tip radius, frequency, and spring constant) took place. To calculate the Young’s modulus of the proteins, the following steps were performed. First, the difference between the cantilever deflections as detected on the hard surface and on the soft protein samples, which describes the deformation of the protein sample under the tip load, was calculated.2 The obtained indentation values were plotted against the force to acquire the indentation versus force curves. The approaching part of the force curve was considered for calculating the indentation. Next, the indentation−force curves were fitted to the Hertz model, which describes the elastic deformation of a soft sample by a rigid indenter.25 Because ferritin is a globular protein, the same method as reported earlier by Parra et al.2 was applied. The Hertz model considering the spherical and conical tip geometry25,26 of the forms (A) δ = [3(1 − ν2)/(4ER1/2)]2/3F2/3 (for a spherical indenter, eq 1) and (B) δ = [π(1 − ν2)/(2E tan α)]1/2F1/2 (for a conical indenter, eq 2) was used, where ν is the Poisson ratio of the sample, which is assumed to be 1/3 as in studies reported by Radmacher et al.1 and Parra et al.2 who used a silicon nitride lever similar to the SNL-10 cantilever used in the present study. Here, δ, F, E, R, and α are the indentation, force, Young’s modulus of the sample, radius of the tip, and half-opening angle of the conical tip, respectively. Current (I)−Voltage (V) Acquisition by Current-Sensing AFS. The I−V measurements on the spin-coated ferritin films were made using CSAFS in contact mode where the electrical characteristics were recorded independent of force feedback. In CSAFS experiments, Ti/Pt coated cantilevers (Micromasch, Estonia) having a spring constant of 2 N/m, a tip radius of ∼35 nm, a length of 110 μm, and a width of 40 μm were used. The conductive probe was initially engaged with the sample at minimal force (in force feedback). The I−V sweeps were made between ±1.5 at 1 V/s (given as a tip bias with respect to the substrate), and the resulting current responses were recorded. The I− V experiments were repeated for ∼100 different locations from different areas as well as from different samples. Each I−V spectrum acquired on a protein molecule was an average over five sweeps. During each measurement, the force set point was calibrated and adjusted as required. To calculate the absolute force at a particular point, the force−distance curves were recorded to relate the force set

(MWCNTs) were reinforced by the formation of hydrogen bonds between ferritin-functionalized MWCNTs and the polymeric films.12 Our present work aims to study the mechanical properties of the ferritin layers on solid substrates by atomic force spectroscopy (AFS). Here we used (AFS), a powerful tool to measure the nanomechanical properties of biological samples13−19 based on the pushing experiments that can provide information on the mechanical properties of the samples through the measurement of approach force curves. Previously, we reported the pressure-stimulated electrical conductance of ferritin proteins by current-sensing AFS where we showed that the electrical conductivity of the ferritin proteins could be modulated by the application of compressive force.20 Within this framework, we will try to establish a correlation between the nanoscale mechanical properties and the pressurestimulated electronic response of the ferritin proteins. Four ferritin systems (i.e., the holoferritin (∼2600 Fe atoms), the two Fe(III)-ferritins (∼750 and ∼1400 Fe atoms), and the apoferritin) will be employed so that any role of the metal content in such correlations can be elicited.

■

MATERIALS AND METHODS

Preparation of Protein Solutions. Equine spleen holoferritin and apoferritin were obtained from Sigma and used as is. The Fe(III)ferritin samples were prepared using a procedure reported by Snow et al.21 (details in Supporting Information). Preparation of Substrates. The preparation of the flameannealed Au(111) substrate, the NHS-EDC-modified Au(111) substrate, and the highly oriented pyrolytic graphite (HOPG) substrate were carried out using standard procedures described in the Supporting Information. Preparation of Protein-Modified Surfaces. Protein modification of gold substrates was undertaken by immersing a bare Au(111) or a modified Au(111) substrate into a 20−40 pM ferritin solution at room temperature (24 ± 1 °C), followed by 12 h of incubation (in the case of bare gold) or 1 h (in the case of modified gold). The gold piece was washed with 0.5 mL of filtered tris buffer solution (0.1 M tris and 0.1 M NaCl, pH 8.0) as in the case of holo-/apoferritins or the MOPS buffer (50 mM MOPS, 0.1 M NaCl, pH 7.5) as in the case of the Fe(III)-ferritins, followed by washing with 1 mL of Milli-Q water and drying gently with a stream of nitrogen gas. For AFM imaging in fluid, the respective buffers (i.e., tris buffer as in the case of holo-/ apoferritins and the MOPS buffer as in the case of the Fe(III)ferritins) were applied. To develop submonolayer deposits of ferritins on an HOPG surface, 10 μL of a 1.5 nM ferritin solution was deposited onto freshly cleaved HOPG, kept in a covered Petri dish for 30 min, washed with 1 mL of the tris buffer (0.1 M tris, 0.1 M NaCl, pH 8.0) and 1 mL of Milli-Q water, and then dried gently under a stream of nitrogen gas. The AFM images were captured in a buffered medium using tris buffer (0.1 M tris, 0.1 M NaCl, pH 8.0). To develop films on the HOPG surface, ferritin solutions of 1.9−2.5 μM concentration were spin-coated at 2800−3000 rpm for 25−30 s onto a freshly cleaved HOPG substrate. After spin-coating, the sample was washed with 0.5 mL of the respective buffer solutions followed by 1 mL of Milli-Q water and dried under a weak stream of nitrogen gas. AFM and AFS Data Acquisition and Analysis. AFM imaging in intermittent contact mode (AAC mode) and force curve acquisition in the contact mode were performed using PicoLE AFM equipment (Agilent Corp., USA) using PicoView 1.12.2 software with a 10 μm × 10 μm scanner at room temperature (24 ± 1 °C). A single cantilever (SNL-10, Veeco) was used for all of the experiments described in this study except for the scratching experiments. The SNL-10 cantilever was calibrated using the Thermal K program that interfaces with the imaging software to calculate the spring constant of a cantilever by the thermal method,22,23 and the calibrated spring constant was found to 12512

dx.doi.org/10.1021/la402522m | Langmuir 2013, 29, 12511−12519

Langmuir

Article

point to the cantilever deflection. All CSAFS measurements were performed under ambient conditions where the temperature and humidity were maintained at 24 ± 1 °C and 35−45%, respectively. The current−voltage measurements were not made in buffer solution in order to avoid extraneous influences of buffer ions on the measurements. In the case of a storage metalloprotein such as ferritin, such a non-physiological condition may be less important than for small functional enzymes or electron-transfer proteins. Also, because the water molecules would be present on the protein exterior, which is the same for all four ferritin systems, this factor could be a common source of error for all of the ferritin samples that we studied in this work. Thus, the presence of moisture would not alter the comparative picture that is elicited from the present study.

linkage formation between the amine groups of the surfaceexposed lysine residues of ferritin and the carboxylic ends of the 3-MPA molecules,27 which are immobilized on the gold surface via strong gold−thiol interactions,28 appeared to be a better strategy than direct immobilization of the ferritin molecules via gold−sulfur interactions.29 Recently, Kim et al. experimentally verified the immobilization of ferritin molecules on gold surfaces via gold−sulfur interactions.30 The ferritin films that we used are likely to be monolayers, as apparent from the results of the contact mode scratching experiments on the films prepared on both modified and bare gold substrates. The observed shapes of ferritin molecules were generally spherical, which conforms well to the respective X-ray structures.31,32 The width values as obtained from intermittent contact AFM imaging are generally found to be less informative as a result of a relatively large tip radius (∼7 nm in the present case) and the tip geometry-induced convolution effects.33 Therefore, the AFM width values were not measured although we verified that the molecules were similarly sized, which indicates that no major aggregation of the ferritins took place upon adsorption. The measured averaged height values for apoferritin and holoferritin molecules, as obtained from the images of the protein films on the bare Au(111) surface, were found to be 4.5 ± 1.1 and 5.2 ± 0.7 nm, respectively. The discrepancy between the theoretical value (∼12 nm)8 and the experimentally determined values can be explained in terms of tip-induced compression of the soft protein exterior of the ferritin molecules, which are directly adsorbed on the hard solid-gold surface. However, when the protein molecules were not directly exposed to the gold surface but were adsorbed onto a layer of 3-MPA, increased height values for both apoferritin and holoferritin molecules (i.e., 8.5 ± 0.9 and 9.0 ± 1.0 nm, respectively) were observed, indicating less tip-induced compression of the ferritin proteins on the modified surface. This observation of increased height values of the proteins on the modified gold surface could also indicate less adsorptioninduced deformation/flattening, which is typically associated with substrate−molecule non-specific electrostatic interactions. Measurement of Young’s Modulus of the Ferritin Proteins. To quantify the mechanical properties of the ferritins, force curve (force vs distance) experiments were executed using a cantilever of calibrated force constant value 0.546 N/m. In Figure 2A, an overlay of the averaged approach force curves obtained for bare Au(111), holoferritin, and apoferritin on bare Au(111) is shown. Noticeably different cantilever deflection behavior in these three cases could be observed, indicating clearly different surface hardness properties. Figure 2B shows the overlay of averaged approach force curves for bare Au(111), modified Au(111), holoferritin, and apoferritin on a modified Au(111) surface. The observation that the distance (or piezo displacement) is shorter (for the same value of the cantilever deflection) for the modified gold substrate than for the bare gold substrate indicates that the indentation of the protein layer by the cantilever is greater for the protein films prepared on the bare gold substrate. The overlay of the averaged approach force curves for holoferritin, the two Fe(III)-ferritin proteins, and apoferritin on the modified gold surface is presented in Figure 2C. Clear differences in the cantilever deflection behavior among the four differently iron-loaded ferritins could be observed. Interestingly, it was found that for a fixed distance the cantilever deflection value for the four ferritin systems was directly proportional to the iron content/molecule. An

■

RESULTS AND DISCUSSION In the present study, the relationship between the mechanical response of ferritins with four different iron contents (apoferritin, ∼750 and ∼1400 iron-containing Fe(III)-ferritins, and ∼2600 iron-containing holoferritin) against an applied force within 2 and/or 10−70 nN (by force curve analysis) and the electrical response of these ferritin systems under pressure stimulation across 17−66 nN (by CSAFS measurements) has been explored. All of the measurements were performed on ferritin film structures to minimize the influence of surface defects on AFM/AFS observations. To determine the role of the substrate in the elastic response of the ferritins, the force curve measurements were made on bare Au(111), 3-MPAmodified Au(111), and freshly cleaved HOPG substrates. In this study, all of the AFM/AFS experiments were carried out in a buffered medium. Formation of Ferritin Film Structures on a Gold Surface. Figure 1 shows the characteristic in situ AFM

Figure 1. AFM topographic images for (A) the 3-MPA-NHS-EDCmodified Au(111) surface, (B) apoferritin, (C) holoferritin, (D) Fe(III)-ferritin (∼1400), and (E) Fe(III)-ferritin (∼750) films on the modified Au(111) surface, taken under buffered conditions. z ranges for (A) 0−4.1, (B) 0−4.5, (C) 0−4.0, (D) 0−6.1, and (E) 0−4.3 nm; the scale bar is 150 nm.

topographic images of the modified Au(111) substrate and the ferritin films on the modified Au(111) substrate. Although the protein molecular contours could be well resolved on the modified Au(111) substrate, the individual molecules could barely be visualized, and streaky features were routinely observed on the bare Au(111) surface (Figure S3 in the Supporting Information), indicating better anchoring of the proteins on the modified gold surface than on the bare gold surface. Evidently, the NHS-EDC-assisted covalent amide 12513

dx.doi.org/10.1021/la402522m | Langmuir 2013, 29, 12511−12519

Langmuir

Article

Figure 2. Overlay of averaged force curves for (A) bare Au(111), holoferritin, and apoferritin on bare Au(111), (B) bare Au(111), modified Au(111), holoferritin, and apoferritin on modified Au(111), and (C) holoferritin, Fe(III)-ferritins (∼750 and ∼1400), and apoferritin on modified Au(111). Indentation vs force curves for (D) apoferritin and holoferritin on bare Au(111) and (E) apoferritin, Fe(III)-ferritins (∼750 and ∼1400), and holoferritin on modified Au(111). Solid lines indicate the fitting of the respective curves following the Hertz model for a spherical indenter, (F) plots of averaged values of the Young’s modulus for four ferritin systems vs no. of iron atoms loaded per ferritin molecule.

have a significant contribution from the compression of the NHS-EDC layer (even though the relative trend would not change), we tried to fit the force−indentation profile for this layer with the Hertz model, but the data could not be well-fitted with the equation. The Young’s modulus values of apoferritin and holoferritin were increased by 41 and 57%, respectively, on the modified gold surface. It is worth mentioning here that the Young’s modulus value of holoferritin was 1.72 times higher than that for apoferritin on the bare Au(111) surface whereas it was 1.91 times higher in case of the modified Au(111) surface. These observations mean that both ferritins were more rigid (or less compressible) on the modified surface, and their elastic behavior could be better discriminated on the modified surface. Figure 2F shows the plots of averaged values of the Young’s modulus of four ferritin systems versus the number of iron atoms loaded per ferritin molecule, which reveals that the extent of metal loading has a direct influence on the elastic behavior of the ferritin systems. Few previous reports on the Young’s modulus of the globular proteins by AFM,1−3 considering spherical tip geometry, are found to match well with our derived Young’s modulus values. The Young’s modulus values that we obtained in the case of conical geometry were expectedly quite different (Table S1 in Supporting Information). Mechanical Response of the Ferritin Systems and Its Relation to the Proteins’ Electronic Response under Compressive Force Loads. To identify a relationship between the mechanical response of ferritins with different iron contents under various force loads and their electron transport behavior with respect to the applied force at the nanoscale, we first measured the indentation values for apoferritin and holoferritin films along with the two Fe(III)ferritins (∼750 and ∼1400) against different applied forces starting from 10 to 70 nN at 10 nN intervals on a modified Au(111) surface (Figure 3A−G). In general, each of the curves obtained in these set of experiments was nonlinear. With increasing applied forces, the indentation values for all four proteins increased monotonously. From the plots of the highest

increasingly greater extent of indentation for apoferritin than for holoferritin as the force value was increased could be observed on both the bare gold and the modified gold surfaces (Figure 2D, E). This probably reflects the fact that the presence or absence of the iron core makes a clearly detectable difference in the indentation profiles. A comparison among the indentation versus force profiles for the four ferritin systems acquired on the modified Au(111) surface (Figure 2E) suggests that the extent of their indentations for the same amount of force applied follows an order that is the inverse of the order of the metal content (i.e., holoferritin (∼2600) > Fe(III)-ferritin (∼1400) > Fe(III)-ferritin (∼750) > apoferritin). The indentation versus force profiles were fitted with the Hertz model, where the fitting could be better performed with eq 1 (spherical geometry) (Figure 2D, E) than with eq 2 (conical geometry) (Figure S4 in Supporting Information). This indicates that the spherical tip geometry is more adequate for explaining our experimental data. The indentation values measured for both apoferritin and holoferritin on a modified surface were smaller than on the bare Au(111) surface, which was reflected in the greater Young’s modulus values obtained in the case of the modified Au(111) surface (Table 1) compared to the values obtained (0.25 ± 0.1 and 0.43 ± 0.1 GPa for apoferritin and holoferritin, respectively) on the bare Au(111) surface. To calculate the Young’s modulus for the 3-MPANHS-EDC layer, because under similar loads, the NHS-EDC and protein layers are compressed to similar extents (Figure 2B), which implies that the calculated Young’s modulus values Table 1. Averaged Values of the Young’s Modulus of the Ferritin Proteins in Case of the Molecules Adsorbed onto Modified Au(111) and Assuming Spherical Tip Geometry ferritins holoferritin Fe(III)-ferritin (∼1400) Fe(III)-ferritin (∼750) apoferritin

Young’s modulus (GPa) 0.68 0.52 0.42 0.35

± ± ± ±

0.2 0.2 0.1 0.1 12514

dx.doi.org/10.1021/la402522m | Langmuir 2013, 29, 12511−12519

Langmuir

Article

Figure 4. Plots of the rate of deformation (nm/nN) values against the applied force loads (nN) for apoferritin, Fe(III)-ferritins (∼750 and ∼1400), and holoferritin.

the rate of deformation for apoferritin increased up to 40 nN of applied force, beyond which it decreased slightly. In contrast, in the case of holoferritin, the rate of deformation decreased from the beginning (i.e., from 10 to 50 nN), after which it became almost constant and increased a little. Similarly, in the case of the Fe(III)-ferritin samples, the rate of deformation decreased from the beginning up to 50 nN (in the case of Fe(III)-ferritin (∼1400)) or 60 nN (in case of Fe(III)-ferritin (∼750)). Beyond these points, the rate of deformation increased for both Fe(III)-ferritins. From these data, we can infer that apoferritin behaves drastically different than the other three ferritin systems, most likely as a result of the major structural difference due to the absence of the iron core. The initial increase in the rate of deformation in the case of apoferritin could be due to the ease with which the AFM probe could indent the protein molecule, which is devoid of the iron core. After a certain threshold value of the atomic packing density was reached, further compression became gradually ineffective and the rate of deformation was reduced. In the case of the iron-containing ferritins, probably the presence of the hard core introduced a general rigidity into whole protein molecules, which is reflected in their tendency to withstand deformation. However, a certain structural change (or loss) is indicated after the application of a large amount of force (beyond 50−60 nN), which allowed deformation to occur more easily, leading to an increase in the rate of deformation. Although in the case of naturally occurring holoferritin, a dense core structure is known to form and a number of reports have been made on the core structure (e.g., that it could be a heterogeneous solid with a dense nucleus and could spread out like tree roots or the rock piles35) and the core formation mechanism (i.e., a nucleation-driven process36,37), in the present context we have no clear experimental evidence that dense iron core structures were formed for Fe(III)-ferritins containing ∼750 and ∼1400 iron atoms. However, because the rate of deformation versus force plots (Figure 4) for these two proteins are more like the plot for holoferritin, we concluded that the protein behavior was related to the iron core and not the protein part alone (as in the case of apoferritin). The observation that the plot for Fe(III)-ferritin containing ∼1400 iron atoms is more similar to the plot for holoferritin than to Fe(III)-ferritin containing ∼750 iron atoms probably indicates that the core structures formed in the two Fe(III)-ferritins are different in nature. The electrical conductance experiments were performed on HOPG because electrical contacts with ferritin molecules

Figure 3. (A−G) Averaged deformation (nm) vs force (nN) curves for apoferritin, Fe(III)-ferritins (∼750 and ∼1400), and holoferritin on the modified Au(111) surface at increasing forces applied starting from 10 to 70 nN. (H) Plots for the highest deformation (nm) vs the respective force value (nN) (with mean deviations shown) for apoferritin, Fe(III)-ferritins (∼750 and ∼1400), and holoferritin on a modified Au(111) surface.

deformation values for the different forces applied (Figure 3H), more or less clear differences could be observed among the four protein systems, although only for the high force values (i.e., 40 nN and above). However, differences observed between apoferritin and Fe(III)-ferritin (∼750) and between holoferritin and Fe(III)-ferritin (∼1400) were rather small. It is worth mentioning here that the deformation induced on protein films under the highest load (i.e., 70 nN) reached values that were higher than the film thickness. This could be related to the nonlinearity of cantilever deflection for high deflection values34 and probably also having contributions from the compression of the underlying 3-MPA layer. From Figure 3A−G, it is evident that the indentation values measured for apoferritin were always greater in comparison to those for holoferritin (i.e., iron-loaded holoferritin molecules were more resistant to the applied forceinduced indentation than were empty apoferritin molecules). From the plots of the rate of deformation with increasing applied force load on each system (Figure 4), it was found that 12515

dx.doi.org/10.1021/la402522m | Langmuir 2013, 29, 12511−12519

Langmuir

Article

adsorbed on the modified Au(111) surface could not be made by a CSAFS tip, and most often I−V responses representing direct contact with a conductive substrate or dielectric breakdown type of behavior were observed. Because it was shown earlier that the I−V responses of ferritins obtained from STS (on the bare Au(111) surface) and from CSAFS (on HOPG surface) experiments were quite similar,20 in the present framework, the correlation between electrical and mechanical properties of ferritins is expected to be realistic. However, we still checked for the deformation behavior of ferritins adsorbed on the HOPG substrate by first forming a sub-monolayer deposit of ferritins onto HOPG, followed by AFS measurements. We worked on the sub-monolayer deposit (Figure 5) so

Figure 6. (A) Overlay of averaged force curves for HOPG, holoferritin, and apoferritin on HOPG. (B) Indentation vs force curves for apoferritin and holoferritin on HOPG. (C) Overlay of averaged force curves and (D) indentation vs force curves for apoferritin on bare Au(111), HOPG, and modified Au(111). (E) Overlay of averaged force curves and (F) indentation vs force curves for holoferritin on bare Au(111), HOPG, and modified Au(111). (B, D, F) Solid lines indicate the fitting of the respective curves following the Hertz model for a spherical indenter.

Figure 5. AFM topographic image (3D view) showing the characteristic sub-monolayer deposit of ferritin proteins on a bare HOPG substrate obtained under buffered condition.

that we could identify if there was a significant effect of the substrate present in the observed indentation behavior. The height of the ferritin molecule adsorbed on a bare HOPG substrate as a submonolayer deposit under buffered condition was found to be 7−9 nm. Lesser cantilever deflection and a greater extent of deformation could be observed in case of the apo protein compared to the holo form (Figure 6A, B), as also observed earlier in case of the modified gold or bare gold substrates (Figure 2). Only small differences could be detected in the cantilever deflection profiles and the indentation profiles of both apoferritin and holoferritin when a comparison was made between the two cases of HOPG substrate and the modified Au(111) substrate (Figure 6C−F). As observed in the case of modified Au(111) earlier (Figure 2E), better fitting of the indentation profiles could be obtained using the equation for spherical tip geometry. The Young’s modulus values obtained for the HOPG substrate (0.60 ± 0.2 and 0.31 ± 0.1 GPa for holoferritin and apoferritin, respectively) were found to be comparable to those obtained for modified Au(111) (Table 1), indicating that there was no major role of the substrate as long as the comparison was made between modified Au(111) and HOPG substrates. However, the behavior was drastically different for bare gold, which is probably to be expected because HOPG is a fundamentally different substrate compared to gold, having considerably less surface energy than that of a gold surface.38,39 For the I−V experiments, the estimated contact area between the CSAFS tip and the protein film was found to be 630 nm2 considering the tip-induced deformation of the ferritin shell to be 3 nm because the average height for single ferritin molecules considering apoferritin as well as the metal-loaded ferritins was

∼9 nm, which is 3 nm less than ferritin’s crystallographic diameter of 12 nm.8 This contact area could contain a maximum of five to six molecules because the area that each ferritin molecule would occupy is 113 nm2. The present I−V measurements can therefore be considered to be close to a molecularly resolved measurement. In the case of pressure-modulated electronic conduction via apoferritin, it was shown earlier that the current values increased more drastically beyond a certain force value,20 in contrast to holoferritin, which exhibited a more steady increase in current with incremental changes (Figure 2 in ref 20). For apoferritin, the junction resistance (R) remained unchanged at 60−65 GΩ for the force range of 20−50 nN. The R value gradually decreased to 45 GΩ for forces >50−60 nN, and it decreased to 20 GΩ for the highest force value of 66 nN, indicating an abrupt increase in current as the force was increased to >60 nN. This could be an indication of the pressure-induced partial collapse of the central cavity of apoferritin. The R values for holoferritin and the Fe(III)ferritins at particular forces were comparable and were within a narrow range: the initial R values at 16−20 nN were 22−23 GΩ, and at 65−66 nN, they were about 2−3.5 GΩ. The ironcontaining ferritins therefore underwent a smaller decrease in resistance values with increased force application, in contrast to apoferritin (Table 2). 12516

dx.doi.org/10.1021/la402522m | Langmuir 2013, 29, 12511−12519

Langmuir

Article

iron-containing ferritin systems at higher applied force loads. The apoferritin system behaved clearly differently because the averaged initial and final band gap energies were found to be 1.48 and 1.23 eV (Figure 7). An overlay of the averaged I−V

Table 2. Averaged Resistance Values (R) Obtained for Four Different Ferritin Systems by Current Sensing AFS ferritins apoferritin Fe-ferritin (∼750) Fe-ferritin (∼1400) holoferritin

R(GΩ) at 16−20 nN R(GΩ) at 65−66 nN

difference (GΩ)

60 23

20 2.2

40 20.8

23

3.1

19.9

22

3.6

18.4

The reversibility of the I−V profiles as a function of applied force was checked, and it was revealed that the I−V behavior was more or less reversible in the lower force regime of 20−35 nN for apoferritin and holoferritin. In this force regime, if the applied force was increased (by an increment of 5−7 nN at each step), then the current value gradually increased, and if the applied force load was decreased in the same manner (by a decrement of 5−7 nN at each step), then the current decreased almost in the same proportion. However, in the higher force regime (>60 nN), generally the current decreased with a decrease in the applied force load, but not in the same proportion compared to the increments observed during the load increase steps. This probably indicates that at a critical pressure the protein molecules undergo a certain degree of loss of structure, though not totally unfolded. Previous conductance studies also suggested that the compressed protein molecules fall short to revert to their original vertical dimensions on reduction of applied force, which means that a certain degree of loss of structure or irreversible protein deformation can take place upon compression.7 No clear reversibility in the behavior of the two Fe(III)-ferritins could be observed, which probably indicates certain structural differences in comparison to the naturally occurring apoferritin and holoferritin. It is likely that a functionally effective protein fold could not be attained in these two “unnatural” cases probably as a result of the fact that a certain threshold number of iron atoms were not present inside these two types of ferritin molecules. We have shown in the previous sections that with increasing applied loads (until 50−60 nN) the indentation values did not increase appreciably in case of the iron-containing ferritins, which clearly revealed the fact that the presence of a hard metal core inside the protein structure played a key role in resisting the force-induced deformation. In the case of pressuremodulated electronic conduction through holoferritin, a stable incremental increase in current with increasing applied force loads was observed (Figure S5 in Supporting Information). The electronic conduction through the Fe(III)-ferritins at different applied force loads was measured, and the band gap values of these systems were found to be comparable to those of holoferritin within the 17−66 nN force range (Figure S5 in the Supporting Information). For the Fe(III)-ferritin (∼750) and Fe(III)-ferritin (∼1400) systems, the averaged initial (for 16− 20 nN force applied) band gap energies were 1.05 and 1.11 eV, respectively, and the final (for 65−66 nN force applied) band gap energies were 0.77 and 0.61 eV, respectively. These values are found to be comparable to the corresponding values obtained for holoferritin that contains 2660 Fe atoms (1.08 eV for the low force situation and 0.81 eV for the high force situation). Although the band gap values at 16−20 nN were very similar, the difference in magnitude of the band gaps increased for the 65−66 nN force applied, indicating better discrimination of the electrical conductivities of these three

Figure 7. Overlay of averaged I−V responses for apoferritin, Fe(III)ferritins (∼750 and ∼1400), and holoferritin obtained for (A) the minimum applied force values (16−20 nN) and (B) the maximum applied force values (65−66 nN). Overlay of averaged conductance curves of the four ferritin systems for (C) the minimum applied force values (16−20 nN) and (D) the maximum applied force values (65− 66 nN). Bias range ±1.5 V.

responses and conductance curves of the four different ferritin systems for minimum (16−20 nN) and maximum (65−66 nN) applied forces is shown in Figure 7. To determine whether the effect of a high force load could reveal any characteristic trend in the conductivities of the ferritins as a function of metal content, the current values were measured for the four ferritin systems at different bias voltages. For example, for the 1.1 V bias, the current values for apoferritin were 0.26 nA for the minimum force and 1.16 nA for the maximum force applied. In the case of the Fe(III)ferritins and holoferritin, the current values were 0.87−0.94 nA for the minimum force situation (Table S2 in Supporting Information), and the current values were 2.23, 2.45, and 2.96 nA, respectively, for the maximum force applied (Table 3). Though the current values for three metal-loaded ferritin systems were comparable to each other for the minimum force range (16−20 nN), the currents measured at 65−66 nN could be well-discriminated and they followed an order that is the Table 3. Averaged Values of Current Obtained for Four Different Ferritin Systems by Current Sensing AFS for the Maximum Forces (65−66 nN) Applied

ferritins apoferritin Fe(III)-ferritin (∼750) Fe(III)-ferritin (∼1400) holoferritin 12517

current values (nA) at 0.7 V

current values (nA) at 0.9 V

current values (nA) at 1.1 V

current values (nA) at 1.3 V

0.15 0.46

0.44 1.02

1.16 2.23

3.26 4.33

0.48

1.11

2.45

5.37

0.66

1.45

2.96

5.43

dx.doi.org/10.1021/la402522m | Langmuir 2013, 29, 12511−12519

Langmuir

■

same as that for the metal content. A similar observation of a direct correlation between the current values at high force situation and the metal content could be made for other bias voltages of 0.7, 0.9, and 1.3 V as well (Table 3). The slopes of the I−V curves (considering the positive side only) were greater for the high-force situation compared to those for the low-force situation for all the four proteins, irrespective of the metal content (Table S3 in the Supporting Information). This observation correlates well with the fact that for the high-force situation the atomic packing density of the protein shell part, which is common in all four cases, becomes high enough to allow a rapid increase in current for the smallest change in potential. That the compression-induced increase in current has an active contribution from the compression of the protein shell of the ferritin proteins is also evident from the observation that the slope values in the case of the metal-free apoferritin were the maximum among the four ferritin systems. We conclude that the extent of metal loading is probably not the sole determining factor in pressure-modulated electronic conduction through the ferritins. Had it been so, the conductance of holoferritin (∼2600 iron atoms) at any applied force load would have been greater than that of the Fe(III)ferritin containing ∼750 iron atoms. In the present study, we have observed that the rates of deformation for both Fe(III)ferritin systems (∼750 and ∼1400) were comparable to those for holoferritin (Figure 4). This could imply that with the addition of metal atoms inside the apoferritin cavity the overall protein structure generally became more rigid, irrespective of the total number of metal atoms. This could happen if the metal atoms were predominantly aligned along the inner walls of the protein shell, rather than forming condensed aggregates alone in a localized manner. An earlier study of the Co(III)ferritin system, which reports the formation of hollow cobalt nanoparticles consisting of approximately 1000−2000 cobalt atoms inside the apoferritin cavity, partially supports this proposition.40 It is apparent that the contribution from the metal part of these proteins in their force-induced conductance profiles arises from the metal atoms that reside along the inner wall of the protein shell, at least for the lower force ranges. Only when a large amount of force is applied and the protein is compressed enough could the metal atoms that reside deeper inside the core be accessed, making the metal content a determining factor of the force-induced conductance profile.

Article

ASSOCIATED CONTENT

S Supporting Information *

Preparative methods, data acquisition/analysis details, UV−vis spectra of ferritin samples, TEM images, AFM images on a bare gold(111) surface, indentation versus force curves with fitting to a conical indenter equation, Young’s modulus values considering a conical indenter, AFM images of ferritin films on HOPG, respective I−V curves across a range of tip−sample forces, current values at low force, and slope values for low and high force situations. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We gratefully acknowledge the financial support from DBT, Government of India (grant no. BT/PR-11765/MED/32/107/ 2009), research fellowships to T.R., S.B., and S.M. from the Indian Association for the Cultivation of Science, Kolkata, and the Council of Scientific and Industrial Research, Government of India, respectively. We also thank Dr. S. Ray of Department of Material Science, IACS, Kolkata, for ICP measurements.

■

REFERENCES

(1) Radmacher, M.; Fritz, M.; Cleveland, J. P.; Walters, D. A.; Hansma, P. K. Imaging adhesion forces and elasticity of lysozyme adsorbed on mica with the atomic force microscope. Langmuir 1994, 10, 3809−3814. (2) Parra, A.; Casero, E.; Lorenzo, E.; Pariente, F.; Vázquez, L. Nanomechanical properties of globular proteins: lactate oxidase. Langmuir 2007, 23, 2747−2754. (3) Afrin, R.; Alam, M. T.; Atsushi, I. Pretransition and progressive softening of bovine carbonic anhydrase II as probed by single molecule atomic force microscopy. Protein Sci. 2005, 14, 1447−1457. (4) Alessandrini, A.; Cornia, S.; Facci, P. Unravelling single metalloprotein electron transfer by scanning probe techniques. Phys. Chem. Chem. Phys. 2006, 8, 4383−4397. (5) Ron, I.; Sepunaru, L.; Itzhakov, S.; Belenkova, T.; Friedman, N.; Pecht, I.; Sheves, M.; Cahen, D. Proteins as electronic materials: electron transport through solid-state protein monolayer junctions. J. Am. Chem. Soc. 2010, 132, 4131−4140. (6) Zhao, J.; Davis, J. J. Force-dependent metalloprotein conductance by conducting probe AFM. Nanotechnology 2003, 14, 1023−1028. (7) Zhao, J.; Davis, J. J.; Sansom, M. S. P.; Hung, A. Exploring the electronic and mechanical properties of protein using conducting atomic force microscopy. J. Am. Chem. Soc. 2004, 126, 5601−5609. (8) Harrison, P. M.; Arosio, P. The ferritins: molecular properties, iron storage function and cellular regulation. Biochim. Biophys. Acta 1996, 1275, 161−203. (9) Levi, S.; Santambrogio, P.; Cozzi, A.; Rovida, E.; Albertini, A.; Yewdall, S. J.; Harrison, P. M.; Arosio, P. Evidence of H- and L-chains have co-operative roles in the iron-uptake mechanism of human ferritin. Biochem. J. 1992, 288, 591−596. (10) Granick, S. Ferritin. I. Physical and chemical properties of horse spleen ferritin. J. Biol. Chem. 1942, 451−461. (11) Shin, M. K.; Kim, S. I.; Kim, S. Reinforcement of polymeric nanofibers by ferritin nanoparticles. J. Appl Phys Lett. 2006, 88, 193901. (12) Bhattacharyya, S.; Sinturel, C.; Salvetat, J. P. Proteinfunctionalized carbon nanotube-polymer composites. Appl. Phys. Lett. 2005, 86, 113104.

■

CONCLUSIONS In the present study, the nanoscale mechano−electronic behavior of four ferritin systems with differently loaded iron atoms (apoferritin, Fe(III)-ferritins (∼750 and ∼1400 iron atoms), and holoferritin (∼2600 iron atoms)) has been investigated against a range of force applied by CSAFS. We show from the measured Young’s modulus values that the amount of iron loaded per ferritin molecule can be directly linked to ferritin’s mechanical response to compressive force. Although in general the force-induced increase in current appears to have a significant contribution from the protein shell part, the contribution of the metal content in observed forceinduced conductance profiles becomes evident when the protein molecule is compressed to a significant extent (here above 65 nN). In the future, it would be interesting to study other ferritin-like proteins and/or nonprotein systems having a metal core surrounded by a soft polymeric envelope in order to assess whether our propositions are generally valid. 12518

dx.doi.org/10.1021/la402522m | Langmuir 2013, 29, 12511−12519

Langmuir

Article

(35) Stanley, H. E., Ostrowsky, N., Eds.; On Growth and Form: Fractal and Non-Fractal Patterns in Physics ; M. Nijhoff: Dordrecht, The Netherlands, 1986. (36) Wade, V. J.; Levi, S.; Arosio, P.; Treffry, A.; Harrison, P. M.; Mann, S. Influence of site-directed modifications on the formation of iron cores in ferritin. J. Mol. Biol. 1991, 221, 1443. (37) Macara, I. G.; Hoy, T. G.; Harrison, P. M. The formation of ferritin from apoferritin, kinetics and mechanism of iron uptake. Biochem. J. 1972, 126, 151. (38) Needs, R. J.; Mansfield, M. Calculations of the surface stress tensor and surface energy of the (111) surfaces of iridium, platinum and gold. J. Phys.: Condens. Matter 1989, 1, 7555−7563. (39) Donnet, J. B.; Brendle, M.; Dhami, T. L.; Bahl, O. P. Plasma treatment effect on the surface energy of carbon and carbon fibers. Carbon 1986, 24, 757−770. (40) Kim, J. W.; Choi, S. H.; Lillehei, P. T.; Chu, S.; King, G. C.; Watt, G. D. Cobalt oxide hollow nanoparticles derived by biotemplating. Chem. Commun. 2005, 4101−4103.

(13) Maivald, P.; Butt, H. J.; Gould, S. A. C.; Prater, C. B.; Drake, B.; Gurley, J. A.; Elings, V. B.; Hansma, P. K. Using force modulation to image surface elasticities with the atomic force microscope. Nanotechnology 1991, 2, 103−106. (14) Tao, N. J.; Lindsay, S. M.; Lees, S. Measuring the microelastic properties of biological material. Biophys. J. 1992, 63, 1165−1169. (15) Vinckier, A.; Semenza, G. Measuring elasticity of biological materials by atomic force microscopy. FEBS Lett. 1998, 430, 12−16. (16) Touhami, A.; Nysten, B.; Dufrene, Y. F. Nanoscale mapping of the elasticity of microbial cells by atomic force microscopy. Langmuir 2003, 19, 4539−4543. (17) Schaer-Zammaretti, P.; Ubbink, J. Imaging of lactic acid bacteria with AFM-elasticity and adhesion maps and their relationship to biological and structural data. Ultramicroscopy 2003, 97, 199−208. (18) Jiao, Y.; Schäffer, T. E. Accurate height and volume measurements on soft samples with the atomic force microscope. Langmuir 2004, 20, 10038−10045. (19) Michel, J. P.; Ivanovska, I. L.; Gibbons, M. M.; Klug, W. S.; Knobler, C. M.; Wuite, G. J. L.; Schmidt, C. F. Nanoindentation studies of full and empty viral capsids and the effects of capsid protein mutations on elasticity and strength. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 6184−6189. (20) Rakshit, T.; Mukhopadhyay, R. Solid-state electron transport in Mn-, Co-, holo-, and Cu-ferritins: force-induced modulation is inversely linked to the protein conductivity. J. Colloid Interface Sci. 2012, 388, 282−292. (21) Snow, C. L.; Martineau, L. N.; Hilton, R. J.; Brown, S.; Farrer, J.; Boerio-Goates, J.; Woodfield, B. F.; Watt, R. K. Ferritin iron mineralization proceeds by different mechanisms in MOPS and imidazole buffers. J. Inorg. Biochem. 2011, 105, 972−977. (22) Zhang, W.; Hughes, J.; Chen, Y. Impacts of hematite nanoparticle exposure on biomechanical, adhesive, and surface electrical properties of escherichia coli cells. Appl. Environ. Microbiol. 2012, 78, 3905−3915. (23) Sullivan, C. J.; Doktycz, M. J. Comparison of the indentation and elasticity of E. coli and its spheroplasts by AFM. Ultramicroscopy 2007, 107, 934−942. (24) Sotres, J.; Barrantes, A.; Arnebrant, T. Friction force spectroscopy as a tool to study the strength and lateral diffusion of protein layers. Langmuir 2011, 27, 9439−9348. (25) Hertz, H. Uber die Berührung fester elastischer Körper. J. Reine Angew. Math. 1882, 92, 156−171. (26) Sneddon, I. N. The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile. Int. J. Eng. Sci. 1965, 3, 47−57. (27) Patel, N.; Davies, M. C.; Heaton, R. J.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. A scanning probe microscopy study of the physisorption and chemisorption of protein molecules onto carboxylate terminated self-assembled monolayers. Appl. Phys. A: Mater. Sci. Process. 1998, 66, S569−S574. (28) Nuzzo, R. G.; Allara, D. L. Adsorption of bifunctional organic disulfides on gold surfaces. J. Am. Chem. Soc. 1983, 105, 4481−4483. (29) Schon, P.; Gorlich, M.; Coenen, M. J. J.; Heus, H. A. Speller, S. Nonspecific protein adsorption at the single molecule level studied by atomic force microscopy. Langmuir 2007, 23, 9921−9923. (30) Kim, J.; Posey, A. E.; Watt, G. D.; Choi, S. H.; Lillehei, P. T. Gold nanoshells assembly on a ferritin protein employed as a biotemplate. J. Nanosci. Nanotechnol. 2010, 10, 1771−1777. (31) Lawson, D. H.; Harrison, P. M. Solving the structure of human H ferritin by genetically engineering intermolecular crystal contacts. Nature 1991, 349, 541−544. (32) Banyard, S. H.; Stammers, D. K.; Harrison, P. M. Electron density map of apoferritin at 2.8-Å resolution. Nature 1978, 271, 282− 284. (33) Markiewicz, P.; Goh, M. C. Simulation of atomic force microscope tip-sample/sample-tip reconstruction. J. Vac. Sci. Technol. 1995, 13, 1115−1118. (34) Ding, W.; Guo, Z.; Ruoff, R. S. Effect of cantilever nonlinearity in nanoscale tensile testing. J. Appl. Phys. 2007, 101, 034316. 12519

dx.doi.org/10.1021/la402522m | Langmuir 2013, 29, 12511−12519