Article pubs.acs.org/Langmuir
How Do Proteins Unfold upon Adsorption on Nanoparticle Surfaces? Hai Pan, Meng Qin, Wei Meng, Yi Cao,* and Wei Wang* National Laboratory of Solid State Microstructure, Department of Physics, Nanjing University, 22 Hankou Road, Nanjing, Jiangsu 210093, PR China S Supporting Information *
ABSTRACT: Owing to their many outstanding features, such as small size, large surface area, and cell penetration ability, nanoparticles have been increasingly used in medicine and biomaterials as drug carriers and diagnostic or therapeutic agents. However, our understanding of the interactions of biological entities, especially proteins, with nanoparticles is far behind the explosive development of nanotechnology. In typical protein−nanoparticle interactions, two processes (i.e., surface binding and conformational changes in proteins) are intermingled with each other and have not yet been quantitatively described. Here, by using a stopped-flow fast mixing technique, we were able to shed light on the kinetics of the adsorption-induced protein unfolding on nanoparticle surfaces in detail. We observed a biphasic denaturation behavior of protein GB1 on latex nanoparticle surfaces. Such kinetics can be adequately described by a fast equilibrium adsorption followed by a slow reversible unfolding of GB1. On the basis of this model, we quantitatively measured all rate constants that are involved in this process, from which the free-energy profile is constructed. This allows us to evaluate the effects of environmental factors, such as pH and ionic strength, on both the adsorption and the conformational change in GB1 on the latex nanoparticle surface. These studies provide a general physical picture of the adsorption-induced unfolding of proteins on nanoparticle surfaces and a quantitative description of the energetics of each transition. We anticipate that it will greatly advance our current understanding of protein−nanoparticle interactions and will be helpful for the rational control of such interactions in biomedical applications.
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few cases.34,41,46,51 Moreover, the kinetic interplay between protein adsorption and protein denaturation for the whole process has not yet been probed. A general picture of adsorption-induced protein denaturation is still missing. The folding and unfolding mechanisms of proteins in solution have been extensively studied using the stoppedflow-based fast mixing technique.56−60 By measuring the apparent reaction rates at different chemical denaturant concentrations, a “chevron” plot is composed, from which the free-energy landscape for the folding/unfolding can be quantitatively measured. By following their folding/unfolding kinetics at various chemical denaturant concentrations, many complex folding/unfolding behaviors have been adequately revealed experimentally, such as the presence of folding intermediate states, parallel folding pathways, and the frustration of the folding energy landscape.59,60 To a certain extent, surface-adsorption-induced denaturation can be considered to be an analog of the unfolding process caused by chemical denaturants: the surfaces of both nanoparticles and chemical denaturants denature the proteins in a concentrationdependent manner. However, nanoparticle-surface-mediated protein denaturation is far more complicated than a simple denaturation process triggered by chemical denaturants because
INTRODUCTION With the rapid development of nanotechnology, nanoparticles have been increasingly used as biosensors, drug carriers, cancer therapeutic agents, and many other biomedical materials.1−7 In all of these applications, interfacial interactions between nanoparticle surfaces and biomacromolecules, especially proteins, are involved.8−11 Thus, it is essential to develop a general understanding of the interactions between proteins and various nanoparticle surfaces.12−18 It is widely accepted that surface adsorption can lead to structural changes in proteins. Such an effect is beneficial for effective surface coating to stabilize nanoparticles and reduce their cytotoxicity using adsorbed proteins because the structural change in proteins leads to the strong adhesion of proteins to various nanoparticle surfaces19−22 and even triggers important biological consequences.23−26 However, this effect could also be a major obstacle for the application of bioactive proteins in medicine and nanotechnology. Because the function of proteins relies on their 3D structures, the conformational changes induced by adsorption may dramatically affect the function of proteins and even result in a loss of biocompatibility, aggregation, and potential immunogenicity.27−31 Therefore, an enormous amount of fundamental research has been conducted to understand protein−nanoparticle surface interactions.32−46 For example, the effects of nanoparticle size, charge, and hydrophobicity on protein adsorption have been studied in great detail.47−55 However, kinetic studies are limited to a very © 2012 American Chemical Society
Received: June 3, 2012 Revised: August 10, 2012 Published: August 13, 2012 12779
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solution with or without proteins was put on a mica surface, which was affixed to a multiple-use AFM sample plate. The sample was left for 30 min to let the latex particles adhere to the mica surface, and then it was washed twice with 50 μL of pure water to remove unabsorbed nanoparticles and salts. The sample plate was covered with a Petri dish to avoid any possible contamination and was left for 0.5−1 h to dry out completely. AFM images were taken in intermittent contact mode using AC-150 sharp silicon cantilevers from Olympus. The typical tip radius of the cantilevers is ∼10 nm, and the resonance frequency is ∼200−500 kHz. Because the size of the nanoparticles measured using AFM could be broadened by the convolution effects arising from the finite size of the AFM tip, the diameters obtained directly from AFM images are typically not accurate. However, when spherical geometries for both the cantilever tip and the sample were assumed, the dimensions of the nanoparticles (Wobs) could be estimated from the measured height using the following equation:67 Wobs = (4hRt)1/2, where h is the sample height and Rt is the average radius of the AFM cantilever tip. Circular Dichroism (CD). The CD spectra were obtained on a Jasco J-815 spectropolarimeter equipped with a Peltier temperature control system set at 25 °C, and the secondary structure was followed in the far-UV regime (200−260 nm) using a protein concentration of ∼0.1 mg mL−1 in water and an optical path of 1 mm. The results were expressed as the mean residue molar ellipticity [θ]. The reported CD spectra were averaged from 10 scans with a response time of 1 s and a spectral bandwidth of 1.0 nm to increase the signal-to-noise ratio. Fluorescence Spectroscopy. All steady-state emission spectra were obtained with a Jasco FP-6500 spectrofluorometer. Tryptophan emission spectra were excited at 285 nm with emission collected from 300 to 400 nm with a 3 nm bandwidth. The background fluorescence from latex nanoparticles has been subtracted in the reported spectra. Stopped Flow. The kinetic measurements were carried out by using a stopped-flow apparatus with an MOS-450 light chamber and an SFM-300 device (Bio-Logic, France). The stopped-flow apparatus was equipped with three electric-motor-controlled syringes filled with protein GB1 stock solution, buffer, and latex nanoparticle solution, respectively. In a typical experiment, the desired amounts of protein GB1 solution, buffer, and nanoparticle solution were pushed by the syringes through a high-density solution (HDS) mixer to a quartz sample chamber. The typical mixing dead time is ∼3 ms. The mixed solution was stopped in the sample chamber to observe the structural change in GB1 upon mixing with latex nanoparticles by monitoring its intrinsic fluorescence excited at 285 nm with a 320 nm cutoff emission filter. The sample chamber was flushed automatically in the next measurement. All traces were fitted with a double-exponential equation (Supporting Information). Obtaining the Apparent Dissociation Constant from the Equilibrium Binding Isotherm. The binding isotherm of GB1 on the latex nanoparticle surface was fitted with the following equation
protein adsorption and denaturation are intermingled with each other (Figure 1). Thermodynamic measurements cannot
Figure 1. Schematic of GB1 adsorption on latex nanoparticles with a two-step reversible model.
provide insight into the detailed mechanism underlying such a nanoparticle-adsorption-induced denaturation process. Here we present the first quantitative measurement of the energetics and kinetics of the nanoparticle-adsorption-induced unfolding of a protein using a stopped-flow-based fast mixing technique. We use well-characterized protein GB1,61−65 the B1 binding domain of Streptococcal protein G,66 as the model protein and polystyrene (latex) nanospheres as the model nanoparticle in this study. By quickly mixing GB1 solution with latex nanoparticles using a stopped-flow mixer, we follow the kinetics of adsorption and denaturation of GB1 on latex nanoparticle surfaces. We found that in this process the structural change in GB1 exhibits biphasic behavior: the fast phase occurs on a subsecond time scale, and the slow phase takes place on a time scale of tens of seconds. Typically, the deviation from singleexponential kinetics suggests the presence of intermediate states. Indeed, our results can be well described by a two-step reversible adsorption−denaturation model (Figure 1). In this model, the adsorption and desorption of proteins reach equilibrium rapidly. Then the adsorbed proteins can undergo a reversible structural change on the nanoparticle surface. There are four rate constants involved in this process: the adsorption rate, kon, the desorption rate, koff, the unfolding rate, ku, and the folding rate, kf. By varying the concentration of nanoparticles, we are able to obtain all four kinetic parameters from our measurements. This allows us to quantify the energetics and infer the mechanism that underlies the adsorption-induced unfolding process. This understanding will eventually help us to make use of beneficial protein−nanoparticle interactions and avoid unwelcomed ones by tuning the energetics through the adjustment of environmental factors such as the pH and ionic strength.
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bound % =
EXPERIMENTAL SECTION
[GB1] + n[latex] + Ked − 2[GB1]
([GB1] + n[latex] + Ked)2 − 4[GB1] × n[latex]
Chemicals. Latex particles (80 nm) supplied as a suspension in water at a concentration of 8% w/v were purchased from IDC, Molecule Probes, Invitrogen. All other chemicals were of reagent grade and were used directly in experiments without further purification. Protein Expression and Purification. The plasmid encoding GB1 gene was kindly provided by Prof. Hongbin Li of the University of British Columbia. GB1 was expressed in BL21 and purified by Ni2+affinity chromatography. The purified polyprotein sample was dialyzed against PBS and kept at 4 °C at a concentration of ∼2 mg mL−1. Dynamic Light Scattering (DLS). The DLS experiments were carried out on a Zetasizer ZS90 (Malvern) with a 633 nm laser source. The latex nanoparticles (0.005%) were incubated with 0.1 mg mL−1 GB1 for 1 day before measurement. All solutions were filtered with a 0.22 μm filter membrane. Atomic Force Microscopy (AFM). Images of the latex nanoparticles were obtained with an atomic force microscope (NanoWizard II, JPK). To prepare the AFM samples, 50 μL of a 0.01% latex particle
2[GB1]
(1)
where bound % is the fraction of bound GB1 in the solution; [GB1] is the initial GB1 molar concentration; [latex] is the initial w/v concentration of latex nanoparticles; n is a prefactor that converts the concentration of latex nanoparticles to the concentration of total available binding sites on latex nanoparticles in solution; and Ked is the apparent dissociation constant. On the basis of this definition, the physical meaning of n is the molar concentration of total available binding sites on the latex nanoparticle surface when the latex nanoparticle concentration is 100% w/v.
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RESULTS AND DISCUSSION Evidence of the Adsorption-Induced Unfolding of GB1 on Latex Nanoparticles. Before the kinetic measurement, we first verified the adsorption of GB1 on latex 12780
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nanoparticles. We mixed GB1 solution with latex nanoparticles in phosphate-buffered saline (PBS, pH 7.4) solution. The mixture was incubated for at least 12 h to allow the systems to reach equilibrium. The size of the latex nanoparticles with or without GB1 was then investigated using dynamic light scattering (DLS). As shown in Figure 2a, the diameter of the
Figure 2. Characterization of GB1 adsorbed latex nanoparticles: (a) DLS profiles of latex nanoparticles with and without adsorbed GB1. (b, c) AFM images of latex nanoparticles with and without adsorbed GB1, respectively.
latex nanoparticles increased slightly from 82.1 ± 5.5 nm (mean ± standard deviation) to 93.4 ± 6.3 nm (mean ± standard error) after mixing with GB1, which is roughly consistent with the size change from the presence of a layer of GB1 on latex nanoparticles. Moreover, we did not observe any populations of large particles (>1000 nm in diameter), indicating that there is no detectable nanoparticle aggregation upon GB1 binding. The size change of latex nanoparticles upon GB1 adsorption was also measured using AFM imaging. As shown in Figure 2b,c, the size of latex nanoparticles increased from 79 ± 3 nm (mean ± standard error, counts = 77) to 83 ± 3 nm (counts = 68) after incubation with GB1. Because of the tip-broadening effect in AFM imaging, the size of the nanoparticles obtained using AFM could not provide an accurate estimation of the size change of the latex nanoparticles upon GB1 adsorption. Nonetheless, these results suggested that GB1 is indeed adsorbed onto the latex nanoparticle surface upon mixing. To obtain more insight into the structural change in GB1 upon adsorption onto a latex surface, we monitored the far-UV circular dichoism (CD) and fluorescence spectra of GB1 before and after adsorption. As presented in Figure 3a, the CD spectrum of native GB1 shows a typical α + β structure with negative peaks in the range of 208 to 222 nm. However, upon adding 0.002% w/v 80 nm latex nanoparticles to the solution, the CD spectrum of GB1 is altered dramatically. We observed the increase in the ellipticity of GB1 upon adsorption onto the latex surface, which indicates that the structure of GB1 is partially denatured. It is worth noting that such a structural change does not lead to a random coil structure of GB1 because we did not observe a decrease in the ellipticity at ∼200 nm.
Figure 3. Structural change in GB1 upon adsorption on latex nanoparticles. (a) CD spectra of GB1 adsorbed on latex nanoparticles of different concentrations. The contribution from pure latex nanoparticles has been subtracted. (b) Fluorescence spectra of GB1 adsorbed on latex nanoparticles of different concentrations. The fluorescence from the latex nanoparticles has been subtracted for each trace. (c) Binding isotherm of GB1 on latex nanoparticle surfaces measured on the basis of the fluorescence quenching. The red line corresponds to the fitting to eq 1. (Inset) The fluorescence intensity of GB1 at 340 nm as a function of the latex nanoparticle concentration.
Moreover, the CD spectrum of adsorbed GB1 does not change upon further incubation, suggesting that the structure of GB1 is quite stable on the latex nanoparticle surfaces although it is different from the native structure. When the nanoparticle concentration increased to 0.005% w/v, we observed a further increase of the ellipticity of GB1 as more GB1 proteins adsorbed onto the latex nanoparticle surface. The structural change in GB1 upon adsorption onto the latex nanoparticle surface is also evident from the fluorescence spectra. As shown in Figure 3b, upon mixing with 80 nm latex nanoparticles, the intensity of the fluorescence decreases significantly and blue shifts slightly from 342 to 332 nm. Such a change in fluorescence is mainly due to the alternation 12781
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of the tertiary structure of GB1, which leads to a shift in the chemical environment adjacent to the tryptophan fluorophore. Moreover, the amplitude of fluorescence change is related to the number of latex nanoparticles added to GB1 solution and reaches the maximum when 0.2% w/v latex nanoparticles are present in the solution. This indicates that the amount of proteins adsorbed is directly proportional to the surface areas of the nanoparticles. By assuming that all GB1 binding events are independent, we obtained the saturated amount of GB1 on latex nanoparticles at a GB1 concentration of 0.03 mg mL−1 to be ∼250 (Figure 3c, Supporting Information). This is significantly smaller than the maximum amount of GB1 that the surface area of a nanoparticle of 80 nm can accommodate (∼5000; Supporting Information). Therefore, the surface density of GB1 is quite low, and the independent binding assumption is valid. Moreover, such a nanoparticle-concentration-dependent quenching of GB1 fluorescence suggests that the adsorption and desorption of GB1 on the latex nanoparticle surface is an equilibrium process and does not lead to a permanent structural change in GB1. Indeed, prolonged incubation does not further decrease the fluorescence of GB1, as shown in Figure S1 in Supporting Information. From the binding isotherm, we estimated that the prefactor, n, is ∼11.7 ± 1.2 M (w/v)−1 and the overall dissociation constant of GB1 from the nanoparticle surface, Ked, is ∼0.069 ± 0.007 M based on eq 1. Increasing the GB1 concentration to 0.15 mg mL−1, which is 5 times the concentration of the rest of the measurements, does not affect the measured n and Ked. This result validates the fact that the fluorescence quenching is indeed due to the increase in GB1 adsorption, not the increase in other optical interference (Figure S2, Supporting Information). It is worth noting that because the adsorption is a twostep process the apparent dissociation constant Ked should be equal to KD × (kf/ku) from the following kinetic measurements. Kinetics of the Adsorption-Induced Unfolding of GB1 on the Latex Nanoparticle Surface. Having confirmed the adsorption of GB1 on latex nanoparticles upon mixing, we then use the tryptophan fluorescence of GB1 as an indicator to follow the adsorption-induced denaturation of GB1 on latex nanoparticles via the stopped-flow ultrafast mixing technique. As shown in Figure 4a, the fluorescence of GB1 drops dramatically upon mixing with 80 nm latex nanoparticles, indicating the quick structural change in GB1 upon adsorption. The amplitude of the overall fluorescence change is directly related to the number of nanoparticles added to GB1 solution, which is consistent with the equilibrium fluorescence measurement. Although the folding/unfolding of GB1 as revealed by chemical denaturation is a two-state process, the structural change in GB1 upon adsorption is more complicated: we found that a single exponential fit cannot adequately describe the fluorescence change in GB1 upon adsorption to or desorption from latex nanoparticles (Figure S3), whereas a doubleexponential equation could fit our experimental data reasonably well (Figure S3). The observed rate constant, k1 obs, for the fast phase varies from ∼10.3 to ∼19.5 s−1 depending on the latex nanoparticle concentration (Figure 4b), but the change in the rate constant for the slow phase, k2 obs, is much shallower (Figure 4c). The deviation from single-exponential kinetics typically suggests that the entire process is not cooperative and there should be an intermediate state involved in the denaturation process. According to the model shown in Figure 1, we are able to obtain the adsorption rate, kon, desorption rate, koff, folding rate, kf, and unfolding rate, ku, quantitatively from
Figure 4. Kinetics of GB1 adsorption on latex nanoparticle surfaces. (a) Typical stopped-flow traces of GB1 adsorption on latex nanoparticles of 0.01, 0.02, 0.03, 0.04, and 0.05% w/v concentrations in a PBS buffer at pH 7.4. Each trace shown is the average of six to nine independent measurements and is fitted with a doubleexponential function (black line). (b) Observed rate of the fast phase (k1 obs) as a function of the latex nanoparticle concentration. Fitting to eq 2 yields an adsorption rate constant, kon, of 17.9 M−1 s−1 and a dissociation rate constant, koff, of 8.20 s−1. The ratio koff/kon is the dissociation constant, KD, of 0.46 M. (c) Observed rate of the slow phase (k2 obs) as a function of the latex nanoparticle concentration. Fitting to eq 3 yields folding (kf) and unfolding (ku) rate constants of GB1 on latex nanoparticle surfaces of 0.38 and 2.24 s−1, respectively.
the observed rate constant as follows (detailed derivation in the Supporting Information)60 k1obs = kon × (n[latex] + [GB1]) + koff k 2obs = k f + 12782
k u × (n[latex] + [GB1]) (koff /kon) + n[latex] + [GB1]
(2)
(3)
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where k1 obs, k2 obs, kon, koff, kf, and ku are as defined above; [latex] and [GB1] represent the w/v concentration of latex nanoparticles and the molar concentration of total GB1 in the solution, respectively. As defined in eq 1, n is a prefactor that converts the concentration of latex nanoparticles to the concentration of total available binding sites on latex nanoparticles in solution. n is estimated to be 11.7 ± 1.2 M (w/v)−1 on the basis of the equilibrium binding data shown in Figure 3c. Fitting the relationship of k1 obs with latex nanoparticle concentration using eq 2 yields kon = 17.9 M−1 s−1 and koff = 8.20 s−1. Therefore, the dissociation constant of GB1 to latex nanoparticles, KD, is 0.46 M. It is worth noting that KD is different from Ked. Ked is the overall equilibrium dissociation constant and equals the dissociation constant of the first adsorption process (KD) multiplied by the equilibrium constant of the second denaturation constant (kf/ku). Using the Arrhenius equation, we estimated that the free-energy barrier for adsorption is 6.5 kcal mol−1 and the free-energy barrier for desorption is 7.0 kcal mol−1 . The adsorbed GB1 on nanoparticle surfaces is approximately 0.5 kcal mol−1 more stable than that free in solution. The stabilization effect upon adsorption is marginal. However, because of the large surface area of the latex nanoparticle, the adsorption can be observed even at a latex nanoparticle concentration of as low as 0.002%. Similarly, the folding and unfolding rates of GB1 on latex nanoparticles can be obtained by fitting the relationship of k2 obs with the latex nanoparticle concentration using eq 3. The folding rate kf is calculated to be 0.38 s−1, and the unfolding rate ku is 2.24 s−1. Both rates are significantly slower than the folding and unfolding of GB1 in solution, indicating that the surface binding increases the free-energy barrier between the folded and the unfolded states of GB1. Moreover, the relative stability of folded and unfolded GB1 is reversed on the latex nanoparticle surface. In solution, the folding rate is 20 000 times faster than the unfolding rate, indicating that the folded state is ∼6 kcal mol−1 more stable than the unfolded state.63 However, on the latex surface, denaturant GB1 is ∼1.0 kcal mol−1 more stable than the folded one. This adequately explains why GB1 could gradually be denatured upon adsorption. Energetics of the Adsorption-Induced Unfolding of GB1 on the Latex Nanoparticle Surface. The detailed energetics underlying the adsorption and denaturation of GB1 on the latex nanoparticle surface are summarized in Figure 5.
The adsorption and desorption of GB1 are in fast equilibrium with the low interconversion free-energy barrier of 6.5 kcal mol−1. Therefore, we infer that the driving forces for the adsorption process are mainly dominated by nonspecific interactions, such as electrostatic and van der Waals interactions. These forces have relatively longer force ranges than hydrophobic interactions and hydrogen bonding. However, these interactions can slightly destabilize GB1, making it prone to unfold on latex nanoparticles. This explains the much higher unfolding rate of GB1 on latex nanoparticle (∼2.24 s−1) than free in solution (∼0.04 s−1).63 Upon unfolding, more hydrophobic residues of GB1 are exposed, which undergo strong hydrophobic interactions with the latex nanoparticle surface of polystyrene. These interactions make the unfolded GB1 stable on the latex nanoparticle surface, and breaking these interactions is a prerequisite to the refolding of GB1. Thus, the free-energy barrier for the refolding of GB1 on the latex nanoparticle surface is almost 7 times higher than that in solution.63 Moreover, because the unfolded GB1 on nanoparticle surfaces is ∼1.0 kcal mol−1 more stable than the folded one on nanoparticle surfaces and ∼2.3 kcal mol−1 more stable than that free in solution, the unfolding of GB1 on nanoparticle surfaces further facilitates the adsorption and makes the overall dissociation constant almost 7 times lower than that in the absence of unfolding. The detailed kinetic parameters for the whole process are summarized in Table 1, and the corresponding parameters for the free-energy landscape are listed in Table S1 and Figure S4. How Do Environmental Conditions Affect the Adsorption and Denaturation of GB1 on Latex Nanoparticles? Such quantitative pictures of the adsorption-induced unfolding of GB1 allow us to study the effects of different environmental conditions on this process. We first study the adsorption-induced unfolding of GB1 by latex nanoparticles at different pH values (Figure 6). The isoelectric point of GB1 is 6.2. Therefore, it is slightly negatively charged at pH 7.4, highly negatively charged at pH 9, and slightly positively charged at pH 5.5. The kinetic parameters at different pH values are summarized in Tables 1 and S1 and Figure 6. Clearly, the repulsive forces between proteins and nanoparticles slow down the adsorption while speeding up the desorption. However, the effects of electrostatic interactions on the unfolding and refolding of proteins on nanoparticle surfaces are not apparent, indicating that they play only a minute role in the unfolding and refolding process. This is consistent with our conclusion that the hydrophobic interactions between proteins and nanoparticles are the driving forces for the adsorption-induced unfolding. We also study the effect of ionic strength on the kinetics and the corresponding free-energy landscape of the nanoparticlebinding-induced unfolding of GB1 (Figures 7 and S5). Because the latex nanoparticles that we used in this study bear negative charges under all three conditions as confirmed by their zeta potentials (Table S2), the electrostatic attraction between GB1 and the latex nanoparticle decreases gradually and becomes negative (repulsion) when [NaCl] increases. The electrostatic interactions of GB1 with nanoparticles are strongly reduced at elevated ionic strength as a result of electrostatic screening effects. Therefore, we expect a decrease in the adsorption rate, kon. As shown in Table 2, kon indeed decreases by ∼30% when the sodium chloride concentration in the solution increases from 70 to 200 mM. Similarly, koff increases by ∼44% because of the reduction in protein−nanoparticle interactions by
Figure 5. Free-energy diagram for the adsorption of GB1 on latex nanoparticles. N and Ns represent the native states of GB1 in solution and on the latex nanoparticle surface, respectively. Us represents partially unfolded GB1 on the nanoparticle surface. T1 and T2 correspond to the transition states for the adsorption and unfolding of GB1, respectively. 12783
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Table 1. Summary of the Kinetic Parameters for the Adsorption of GB1 on Latex Nanoparticle Surfaces at Different pH Values in the Presence of 137 mM NaCl pH value
kon (M−1 s−1)
koff (s−1)
KD (M)
kf (s−1)
ku (s−1)
5.5 7.4 9.0
24.5 ± 1.8 17.9 ± 1.1 7.3 ± 0.6
6.7 ± 0.7 8.2 ± 0.4 9.1 ± 0.3
0.27 ± 0.05 0.46 ± 0.05 1.25 ± 0.14
0.26 ± 0.04 0.38 ± 0.08 0.72 ± 0.05
1.38 ± 0.07 2.24 ± 0.18 1.38 ± 0.23
nanoparticle surface are also changed slightly by the ionic strength, further confirming that the electrostatic interactions play only minor roles in the folding/unfolding process (Tables 2 and S3 and Figures 7 and S5). Recent simulation and experimental work suggests that hydrophobic interactions are the major driving force for the adsorption of proteins or peptides on hydrophobic surfaces, such as graphite or carbon nanotubes.21,68,69 However, our experimental results at different pH values and salt concentrations suggest that electrostatic interactions and van der Waals interactions play important roles in the initial adsorption process. Becauase the adsorption process is the prerequisite for the denaturation step, it is also possible to modify the surfaces of proteins and nanoparticles (e.g., linking with poly(ethylene glycol) or charged polypeptide tags) to prevent the initial step of adsorption to nanoparticles, which avoids surface-induced protein denaturation. Although the poly(ethylene glycol) coating has been widely applied to stabilize nanoparticles and proteins for medical applications,70−73 the physical mechanism underlining these strategies is somehow still not well understood. Our results and kinetic model could provide a framework for evaluating the effects of different protein or nanoparticle surface modification techniques and bring about new insights into the rational control of protein−nanoparticle interactions. Many studies show that when nanoparticles get into living bodies they can recruit many proteins from the biological environment to form a layer of protein “corona” outside the nanoparticles.14,18,26,32,36,38 It is the properties of this protein corona that dictate the distribution and biological consequences of the nanoparticles instead of the properties of the nanoparticles per se. Therefore, understanding how proteins interact with nanoparticles to form such a corona is essential. Our results may suggest that the formation of the protein corona is determined not only by the strength of the interactions between proteins and nanoparticles but also by the adsorption kinetics. Because there are many different kinds of proteins in a biological environment, their binding to nanoparticles is a competition process. Whether a protein can eventually bind to nanoparticles is determined by how fast it can adsorb to nanoparticle surfaces. As in a biological environment, the protein concentrations are much higher than the nanoparticle concentrations. Once the nanoparticle surface is occupied, it inhibits the adsorption of other proteins onto the nanoparticle surface. Moreover, the dissociation rate of proteins from nanoparticles is relatively slow because of the unfolding of the adsorbed proteins on the nanoparticle surface. Therefore, the proteins with slower kon will have less chance to occupy the nanoparticle surface, and the adsorption can be considered to be a kinetically determined process. Our method provides a tool for evaluating the adsorption kinetics of proteins to nanoparticles directly and will be helpful in elucidating the formation mechanism of the protein corona in vivo.
Figure 6. Observed k1 obs and k2 obs for the adsorption process of GB1 at different latex nanoparticle concentrations at (a, b) pH 5.5 and (c, d) pH 9.0. The rate constants obtained by fitting with eq 2 for k1 obs and eq 3 for k2 obs are listed in Table 1.
Figure 7. Observed k1 obs and k2 obs for the adsorption process of GB1 at different latex nanoparticle concentrations at NaCl concentrations of (a, b) 70 and (c, d) 200 mM. The rate constants obtained by fitting with eq 2 for k1 obs and eq 3 for k2 obs are listed in Table 2.
increasing the ionic strength of the solution. However, the effects of the ionic strength are not as significant as those of the pH. The folding and unfolding rates of GB1 on the 12784
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Table 2. Summary of the Kinetic Parameters for the Adsorption of GB1 on Latex Nanoparticle Surfaces at Different Sodium Chloride Concentrations at pH 7.4 [NaCl] (mM)
kon (M−1s−1)
koff (s−1)
KD (M)
kf (s−1)
ku (s−1)
70 137 200
19.9 ± 2.4 17.9 ± 1.1 14.1 ± 0.8
6.6 ± 0.9 8.2 ± 0.4 9.5 ± 0.3
0.33 ± 0.08 0.46 ± 0.05 0.67 ± 0.06
0.42 ± 0.14 0.38 ± 0.08 0.35 ± 0.06
1.58 ± 0.29 2.24 ± 0.18 1.93 ± 0.18
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CONCLUSIONS In this work, we provide a quantitative measurement of the kinetics and energetics of the nanoparticle-adsorption-induced unfolding for a protein using a stopped-flow-based technique. This is essential to understanding the mechanism underlying the surface-binding-induced unfolding process. We have also shown how this process can be controlled by external factors such as the pH and ionic strength. These results will be helpful for the control and modulation of protein−surface interactions in nanotechnology and materials science as well as for the development of methods to avoid unwelcome protein denaturation on various nanoparticle surfaces in biotechnology and medicinal chemistry. Although we are currently unable to identify which parts of proteins attach to nanoparticles and lead to the adsorption and denaturation, we propose that, in combination with site-directed mutagenesis, pinpointing the adsorption “hot spot” of the proteins should become possible. Such knowledge will be important for protein engineering and nanoparticle design. Eventually, this will tremendously revolutionize many nanotechnologies, such as protein drug delivery (e.g., insulin), protein coating, and surface-based protein sensor techniques.
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ASSOCIATED CONTENT
S Supporting Information *
Additional experimental methods and data analysis. This material is available free of charge via the Internet at http:// pubs.acs.org.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected];
[email protected]. Author Contributions
This article was written through the contributions of all authors. All authors have given approval to the final version of the article. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Prof. Hongbin Li for providing plasmid encoding protein GB1. This work was supported by the National Natural Science Foundation of China under grant nos. 11074115, 10904064, 91127026, 11104132, and 31170813, the program for New Century Excellent Talents in University, and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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ABBREVIATIONS AFM, atomic force microscopy; CD, circular dichroism; DLS, dynamic light scattering; NP, nanoparticle; PBS, phosphatebuffered saline 12785
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