pubs.acs.org/NanoLett
Protein-Nanoparticle Interaction: Identification of the Ubiquitin-Gold Nanoparticle Interaction Site Luigi Calzolai, Fabio Franchini, Douglas Gilliland, and Franc¸ois Rossi* European Commission, Joint Research Centre, Institute for Health and Consumer Protection, I-21027, Ispra, Varese, Italy ABSTRACT We demonstrate that it is possible to identify the protein-nanoparticle interaction site at amino acid scale in solution. Using NMR, chemical shift perturbation analysis, and dynamic light scattering we have identified a specific domain of human ubiquitin that interacts with gold nanoparticles. This method allows a detailed structural analysis of proteins absorbed onto surfaces of nanoparticles in physiological conditions and it will provide much needed experimental data for better modeling and prediction of protein-nanoparticle interactions. KEYWORDS Gold nanoparticles, NMR, protein-nanoparticle interactions, chemical shift mapping
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anotechnology is expected to have a large socioeconomic impact in practically all industrial activity fields. The use of nanomaterials is constantly increasing in all industrial sectors, in particular in biomedicine with applications in diagnostics and therapeutics.1,2 At the moment not much is known about the effects of nanoscale objects on biological systems and their potential toxicity but recently the interaction of nanoparticles with proteins has emerged as a key parameter in nanomedicine and nanotoxicology.3 When nanoparticles (NPs) interact with proteins, they might alter protein conformation, expose new epitopes on the protein surface, or perturb the normal protein function, which could induce unexpected biological reactions and lead to toxicity.4 Up to now the information about protein structural changes upon binding to nanoparticles has been based mainly on infrared spectroscopy, circular dichroism, fluorescence, and other methods that can monitor changes in the secondary structure of proteins5 but give limited information at the amino acid level. NMR can provide highresolution information on protein structural changes in protein-NP complexes in aqueous solution, thus in an environment very close to that found in physiological conditions. Also, NMR is particularly well suited to detect detailed information in the case of weak protein-target interactions.6 NMR has been used to characterize the interaction between nanoparticles and proteins using either hydrogen/ deuterium exchange experiments7 or by two-dimensional HSQC experiments with 15N-labeled proteins.3 Chemical shift perturbation (CSP) is a well-established technique used to monitor protein-protein interaction and protein-ligand interactions,8,9 and it can be used to identify the protein
domain involved in protein-protein interaction in large supramolecular complexes.10 In CSP, two-dimensional 1 H-15N NMR experiments are used to detect changes in the chemical environment of all coupled H and N atoms of the protein upon formation of protein complexes. We tested whether NMR and CSP could provide information at the atomic level about the interaction of nanoparticles with proteins by analyzing the interaction of ubiquitin protein with gold nanoparticles. Human ubiquitin (hUbq) is a highly conserved regulatory protein containing 76 amino acids that is widely expressed in eukaryotes. Ubiquitin has a very important biological function in the fact that its attachment to proteins “flags” them for degradation by the proteasome. In addition, its relatively small size and the wellcharacterized three-dimensional structure (both in the solid state and in solution) make it a good target for the analysis at the atomic level of its interaction with NP. The secondary structure of ubiquitin contains a long R helix, a short piece of 3(10)-helix, and a mixed beta-sheet with five strands.11 Gold nanoparticles (AuNP) were produced by NaBH4 reduction of a starting solution of sodium citrate and HAuCl4 in water following a standard protocol12 with minor modifications (see Supporting Informations) to provide AuNP in the 10-30 nm size range. The particle size distribution (PSD) of the resulting nanoparticles was measured by dynamic light scattering (DLS). DLS measurements of free, freshly synthesized nanoparticles show that AuNP have near-monodispersed PSD with a mean hydrodynamic diameter of 12.0 nm and polydispersivity index of 0.1 (Figure 1). Upon addition of human ubiquitin the PSD of the AuNP-hUbq complex continues to show the presence of a near-monodispersed sample but now with a mean hydrodynamic diameter of 17.1 nm and polydispersivity index of 0.21 (Figure 1). This indicates that AuNPs are stable in the presence of ubiquitin at pH 7.7 and the addition of protein
* To whom correspondence should be addressed,
[email protected]. Received for review: 05/18/2010 Published on Web: 07/19/2010 © 2010 American Chemical Society
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FIGURE 1. Particle size distribution of free AuNPs (continuous line) at pH 7.7 and AuNPs plus hUbq (dotted line) at pH 7.7. Free AuNPs are near monodispersed with mean hydrodynamic diameter of 12.0 ( 0.2 nm; the addition of human ubiquitin, 50 µM, causes the formation of a stable particle with mean hydrodynamic diameter of 17.1 ( 0.2 nm.
FIGURE 2. Section of 2D [15N-1H]-HSQC NMR spectra of free hUbq (red) and hUbq plus AuNP (yellow) samples, both at pH 7.7, showing chemical shift perturbation for some NH groups upon interaction with AuNP. Black arrows indicate NH backbone groups with the largest chemical shift perturbations (Q2, L15, and E18) in this spectral window.
does not cause the formation of nanoparticles aggregates. The increase in hydrodynamic diameter upon hUbq addition indicates that hUbq molecules interact with the gold nanoparticles in solution. The direct interaction of hUbq with AuNP is confirmed by the shift in the plasmon resonance band of AuNP upon addition of hUbq. The UV-vis spectrum of free AuNP shows a typical intense plasmon resonance band centered at 520 nm. After addition of human ubiquitin this band shifts to 525 nm (see Figure S2 in Supporting Information). This red shift of 5 nm in the surface plasmon resonance band is in agreement with results obtained on a similar system where azurin (a protein with size similar to ubiquitin) interacts with gold nanoparticles of 20 nm.13 Scanning electron microscopy images of the inorganic gold cores of both AuNP and AuNP-hUbq show that the particles do not aggregate and are well monodispersed (see Figure S3 in Supporting Information). Comparing the diameter of free AuNP and AuNP-hUbq complex gives an indication of how many layers of protein are on the surface of gold nanoparticles. The difference © 2010 American Chemical Society
between the mean diameters of free AuNP and AuNPhUbq complex is approximately 5 nm which can be thought as twice as the apparent thickness of the shell coating the AuNP. This number is in good agreement with the expected diameter of the hUbq protein of around 2 nm.14 The direct interaction of hUbq with AuNP is further confirmed by changes in chemical shift of some peaks of the [15N-1H]-HSQC NMR spectrum of hUbq upon addition of AuNP (see Figure 2). Each one of these peaks represents a NH chemical group mainly coming from the NH backbone of each amino acid, and its chemical shifts are very sensitive to the chemical environment. The fact that addition of AuNP changes the position of only some peaks and that the majority are not affected indicates that while the protein interacts with the nanoparticles this interaction is limited to only a few amino acids in the protein and thus is specific. The increase of 5 nm to the mean hydrodynamic diameter of the AuNP-hUbq complex compared to the free AuNP and the changes in the [15N-1H]-HSQC NMR spectrum caused by the addition of AuNP to hUbq indicate that hUbq 3102
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FIGURE 3. Chemical shift perturbation (CSP) of hUbq upon interaction with AuNP for each amino acid present in hUbq. CSP has been calculated with the following formula: CSP ) [(∆1H)2 - (∆15N/5)2]1/2/2; where ∆1H is the difference in chemical shift between free protein and bound protein for each amino acid. Amino acids that show significant CSP upon interaction with AuNP are labeled. For some amino acids there are no data available due to the fact that their peaks are not detect in the HSQC of AuNP-hUbq sample (peptide fragment L8-T12) probably due to flexibility of the loop, while for P19, P37, and P38 there is no backbone NH.
molecules form a monolayer of proteins around the surface of gold nanoparticles. The formation of a protein monolayer of human serum albumin around FePt nanoparticles has also been recently demonstrated by using fluorescence correlation spectroscopy.15 Using simple steric considerations, it is possible to estimate the maximum number of ubiquitin molecules (NMax) on the surface of each spherical gold nanoparticle.16
show significant changes in chemical shift upon addition of AuNP, suggesting that the interaction of ubiquitin with gold nanoparticles is specific and involves only a limited part of the protein. The chemical shift perturbation for each amide NH group of the protein backbone is shown in Figure 3. It has been possible to calculate the CSP for all the amino acids except for the peptide fragment Leu8-Thr12 and for Met1, Arg74, and Gly75 at the N- and C-terminal ends. The peptide fragment Leu8-Thr12 corresponds to a loop region that shows conformational flexibility in the free protein (as evidenced, for example, by the weak signal of Thr12, Figure 2). The inability to detect the NMR signal of the NH atoms for the peptide fragment Leu8-Thr12 is probably due to the increase in conformational flexibility of the loop in the complex. The ubiquitin proteins in the hUbq-AuNP sample are in excess with respect to the nanoparticle molecules; thus the HSQC spectra of hUbq (Figure S1 in Supporting Information) is an equilibrium of free and bound proteins. The fact that there is basically no line broadening of the peaks between free hUbq and hUbq-AuNP indicates that the two species (free and bound) are in fast exchange on the NMR time scale.18 In these conditions the chemical shifts are a weighted average of the free and bound protein ones and thus changes in the chemical shifts of the hUbq-AuNP system are directly correlated to the interaction of the protein with AuNP. The large majority of NH groups have CSP smaller than 0.02 ppm in the presence of AuNP, while there are two polypeptide fragments (comprising Gln2, Ile3 and Leu15, Val17, Glu18) that show substantial changes. The fact that around 90% of the amino acids of the protein show CSP smaller than 0.02 ppm when interacting with AuNP indicates that the interacting protein is folded and undergoes only minor structural changes. The two peptide fragments that show the biggest chemical shift changes are not contiguous
NMax ) 0.65(Rcomplex3 - RAuNP3)/RUbq3
where Rcomplex is the radius of the AuNP-hUbq complex, RAuNP is the radius of AuNP, and RUbq is the radius of hUbq. The above calculation gives an estimated theoretical maximum number of ≈140 protein molecules per nanoparticle. The actual number of hUbq molecules per nanoparticle is most likely lower as the calculation assumes a close packing of proteins on the AuNP surface. On this basis, and under the experimental conditions applied here, there is an excess of protein molecules in solution relative to protein molecules interacting with AuNP. Therefore, the AuNP-hUbq complex can be thought of as a 12 nm sized gold nanoparticle with a 2.5 nm shell composed of less than 140 human ubiquitin molecules around it, surrounded by an excess of free hUbq molecules. To characterize at atomic level the interaction of AuNP with hUbq, we used high-resolution NMR to obtain structural information at the amino acid level about the site of interaction of gold nanoparticle on the protein. The two-dimensional [15N-1H]-HSQC spectrum of human ubiquitin in the presence of AuNP shows a very good spectral resolution indicative of a properly folded protein17 (see Figure S1 in Supporting Information). Figure 2 shows a portion of the superposition of two-dimensional HSQC spectra of free hUbq and AuNP-hUbq samples. Only a few NH backbone groups © 2010 American Chemical Society
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FIGURE 5. Model for the absorption of human ubiquitin to AuNP. Ubiquitin molecules interact with gold nanoparticle with a specific protein surface formed by amino acid residues 2-3 and 15-18 (red). Free protein molecules are in fast exchange (green arrows) with bound protein molecules. Relative sizes of AuNP and ubiquitin molecules are not on scale.
All these data suggest a model for the absorption of the protein to the particles. In this model, depicted in Figure 5, ubiquitin molecules interact with gold nanoparticles via a specific protein surface formed by amino acid residues 2-3 and 15-18 (in red in Figure 5). These molecules form a protein monolayer around gold nanoparticles and are in fast exchange (green arrows in Figure 5) with free protein molecules that are in excess relative to AuNPs. A similar experimental approach for the identification of the protein-NP interaction site can be easily extended to other kinds of nanoparticles and to other proteins. This method has several advantages in the use of low quantities of sample and the fact that it works in solution and is compatible with the use of a large range of different pH values and salt conditions, so in conditions very similar to physiological ones and thus very relevant for the understanding of nanoparticle toxicity in biological systems. The use of modern NMR instruments equipped with a cryoprobe requires very low quantities of 15N-labeled proteins, as low as 0.05 mg of 15N -labeled hUbq per sample in the experiments described here. Thus with limited amounts of samples it will be possible to test the influence of different experimental conditions such as NP size, pH, and ionic strength, on protein-NP interaction. The identification of the site of interaction of different NP on a variety of proteins will also provide much needed experimental data for the modeling of protein-NP interactions. The prediction of the interaction of protein with solid surfaces has recently received large attention with methods using either empirical energy functions19 or ab initio calculations20 and the availability of direct experimental data could greatly improve the accuracy of the
FIGURE 4. (a) Cartoon structure of human ubiquitin. In red are indicated the backbone traces of amino acids that form the AuNPbinding domain: Gln2, Ile3, Leu15, Val17, and Glu18. (b) Electrostatic surface potential of human ubiquitin at pH 7.7. The red and blue colored areas correspond to negatively and positively charged areas, respectively; the green transparency is showing the trace of the protein backbone.
in the amino acid sequence, but they are close in space in the three-dimensional structure of the protein (Figure 4a). Thus the fragment Gln2-Ile3 and Leu15-Glu18 form a domain on the protein surface that interacts with AuNP. The AuNP-binding domain (Gln2-Ile3, and Leu15-Asp18) is at the N-terminal end of the protein and is part of the β-turn-β motif (Figure 4a). To better understand the hUbq-AuNP interaction the surface electrostatic potential of native hUbq at pH 7.7 was calculated (Figure 4b). Human ubiquitin has an isoelectric point of 6.8 and at pH 7.7 is slightly negatively charged, but the charge distribution is not homogeneous with some positive and (more) negative patches on the protein surface. The AuNP-interaction domain is part of the β-turn-β motif at the N-terminal end and is slightly negatively charged but overall it is not the most negatively charged part of the protein. It could be expected that the interaction of ubiquitin with AuNP to be driven by electrostatic interaction with the hUbq molecules somehow displacing the citrate molecules that stabilize AuNP in solution. The experimental data suggest that electrostatic interaction plays a role in the interaction with AuNP but also other factors seem to have a role. For example it will be interesting to see if the inherent mobility of the N-terminal end or the flexibility of the loop in the β-turn-β motif plays a role in the binding to AuNP. © 2010 American Chemical Society
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predicted models in a similar way to what is happening to the modeling of protein-protein interactions.21 One of the major drawbacks of NMR is the size limits of proteins that can be measured: protein of up to 50 kDa are quite standard nowadays and systems of up to 900 kDa22 are accessible with appropriate labeling and pulse sequences.22,23 In summary this study shows that the interaction of ubiquitin with gold nanoparticles is very specific and that using NMR it is possible to identify the specific protein domain that interacts with AuNP. The approach that we have applied to human ubiquitin should be broadly applicable to other protein-NP systems for proteins smaller than 50-60 kDa. The availability of experimental data on the proteinnanoparticle interaction domain will allow a deeper understanding of the structural features responsible for proteinnanoparticle binding and also open up the possibility of better modeling and predicting the interaction of proteins and other biomolecules to nanoparticles.
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Acknowledgment. We thank Dr. Ce´sar Pascual Garcia for acquiring the scanning electron microscopy images of AuNP and Dr. Luca Varani for useful discussions.
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Supporting Information Available. Procedures for preparing gold nanoparticles, ubiquitin-AuNP complex characterization (DLS, NMR), analysis of structural data for the complex, explanation of eq 1, and Figure S1, Figure S2, and Figure S3. This material is available free of charge via the Internet at http://pubs.acs.org.
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