Gold Nanoparticles as Nucleation-Inducing Reagents for Protein

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Gold Nanoparticles as Nucleation-Inducing Reagents for Protein Crystallization Sanga Ko, Hye Young Kim, Inhee Choi, and Jungwoo Choe Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b01346 • Publication Date (Web): 30 Dec 2016 Downloaded from http://pubs.acs.org on January 7, 2017

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Crystal Growth & Design

Gold Nanoparticles as Nucleation-Inducing Reagents for Protein Crystallization Sanga Ko,† Hye Young Kim,† Inhee Choi,* and Jungwoo Choe* Department of Life Science, University of Seoul, Seoul 130-743, Republic of Korea

*E-mail: [email protected] (J.C.) and [email protected] (I.C.)

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ABSTRACT Protein crystallization is a necessary, but time-consuming and difficult step in structure determination by crystallography. The rate-limiting step of crystallization is nucleation, where protein monomers in solution cluster in an ordered fashion to form a stable nucleus. Here, we propose the use of gold nanoparticles to accelerate nucleation and enhance crystal formation. We tested whether gold nanoparticles can facilitate nucleation by interacting with target proteins and inducing favorable clustering. We used differently sized gold nanospheres and gold nanostars to crystallize hen egg-white lysozyme. Our results indicated that gold nanoparticles significantly increased the number of crystallization conditions (by about 20 %). Spherical and larger gold particles were found to be more efficient. Furthermore, the use of gold nanoparticles did not have any adverse effect on data collection or structure determination. Our findings indicate that the use of nanoparticles as protein nucleationinducing reagents can greatly accelerate structure determination by X-ray crystallography.


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INTRODUCTION X-ray crystallography has been the major method to determine the structures of biological macromolecules, and about 89 % of known structures have been determined by X-ray crystallography.1-3 Protein structure determination by X-ray crystallography includes overexpression and purification of proteins, crystallization, data collection, and structure determination.4-5 Among these, crystallization remains the most critical and time-consuming step.6 Protein crystallization requires supersaturation of a protein solution by either increasing the protein concentration or using chemical reagents (also called precipitants) to reduce the solubility of the protein.7 Protein and precipitant concentrations have to be optimized to induce protein nucleation. A concentration too high leads to amorphous precipitation, whereas that too low to a clear solution as a metastable state. Protein crystallization depends on many factors, including the precipitant, pH, temperature, and gravity.8 The effects of these factors have been studied on many proteins. However, ideal crystallization conditions are different for each protein and establishing the optimal conditions for crystallization remains a trial-and-error process. In this regard, induction of protein crystallization using gold nanostructures including spherical gold nanoparticles9-10 and nanoporous gold nucleants11 have been reported. Benificial effects of other nanomaterials such as platinum nanoparticles12 and reductively PEGylated carbon nanomaterials13 on crystallization were demonstrated before. A computational study on the effect of small ‘seed’ particles on the nucleation of spherical particles also showed that seed particles can act as crystallization ‘catalysts’ by promoting nucleation.14 Here, we report a method to increase the success rate of crystallization, using gold nanoparticles of controlled size and shape as a nucleation-inducing reagent. Gold nanoparticles of different sizes and shapes can present diverse surfaces of different curvature 3 ACS Paragon Plus Environment


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and roughness inducing an optimal clustering and packing of proteins for nucleation and crystal growth. Sphere- and star-shaped gold nanoparticles of varying sizes were applied to the crystallization of hen egg-white lysozyme, as described in Scheme 1. The results showed a significant increase in the number of additional crystallization conditions in the presence of gold nanoparticles, and indicated the possible role of gold nanoparticles as a nucleationinducing reagent for protein crystallization. We also collected X-ray diffraction data and determined the structures of lysozyme in the absence and presence of gold nanoparticles to show that gold nanoparticles do not either interfere with structure determination process or cause significant changes in the final structures.

Scheme 1. Schematic illustration of nanoparticle-assisted protein crystallization process. (a) Strategy of conjugation of lysozyme to gold nanoparticles for forming nucleation cores. (b) Nanoparticle-assisted protein crystallization process and the experimental set-up (sitting-drop vapor diffusion method). 4 ACS Paragon Plus Environment

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EXPERIMENTAL SECTION Materials. Lysozyme from chicken egg white (L2879) was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Lysozyme was dissolved in 20 mM sodium acetate buffer (pH 4.5), containing 50 mM NaCl to a concentration of 7.22 mg/mL (470 µM) and diluted by deionized (DI) water (ca. 18 MΩ) in all experiments. Bovine serum albumin (A2153) was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA) and concentrated to 19.3 mg/mL in 20 mM TrisHCl pH 7.5 and 250 mM NaCl. All gold nanospheres (e.g. 5, 10, 20, 50, and 100 nm) were purchased from BBI solutions (Cardiff, Wales, UK). Glycerol was purchased from Samchun Chemical Co. (Pyeongtaek, Gyeonggi-do, Korea). Gold ( Ⅲ ) chloride trihydrate (HAuCl4), hydroquinone, silver nitrate (AgNO3), sodium citrate tribasic dihydrate (HOC(COONa)(CH2COONa)2·2H2O), and 3-aminopropyltriethoxysilane (APTES, 99 %, H2N(CH2)3Si(OC2H5)3) were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Nickel (Ⅱ) chloride hexahydrate solution (NiCl2•6H2O) was purchased from Biopure Co. (Cambridge, MA, USA). MCSG 2T crystallization suite was purchased from Anatrace Co. (Maumee, OH, USA). Preparation of gold nanostars. Gold nanostars (e.g., 15, 30, 60, 125, and 230 nm) were synthesized by on the seed-mediated overgrowth protocol described in a previous report.15 Briefly, 15, 30, 60, 125, and 230 nm gold nanostars were respectively synthesized using 5, 10, 20, 50, and 100 nm gold nanospheres as seeds. First, 300 mL seed solution in 9.8 mL DI water and 1.0 mL of glycerol was stirred at 700 rpm with a magnetic bar. A mixture of 22 mL of 1 % sodium citrate solution, 100 mL of 1 % HAuCl4 solution, and 42.5 mL of 0.1 % AgNO3 solution was rapidly added to the above solution after 5 minutes, immediately followed by the addition of 100 mL of 1 % hydroquinone solution. The color of the reaction



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solution changed from slightly red to deep blue within a few minutes. All solutions were prepared with DI water. Concentrations are specified as weight per volume (w/v). Transmission electron microscopy. For directly confirming the conjugation of lysozyme to the gold nanoparticles, each 500 µL of the diluted gold nanoparticle solutions was sequentially treated with 0.5 µL of 2 mM Ni ions solution and 55 µL of 10 µM lysozyme. Next, 1 µL of the lysozyme-conjugated gold nanoparticle solutions was dropped onto TEM grids (300 mesh, Cu, Ted Pella. Inc., Redding, CA, USA). After 3 minutes, the TEM grid was washed with DI water (ca. 18 MΩ) and dried at room temperature. Transmission Electron Microscope II (JEM-2100, JEOL Ltd., Akishima, Tokyo, Japan) equipped with CCD camera (ORIUS-SC600, Gatan Inc., Pleasanton, CA, USA) was used for imaging. During imaging, the accelerating voltage was 200 kV. Characterization

of

lysozyme-conjugated

gold

nanoparticles

via

dark-field

spectroscopy. To measure the spectral shifts of Rayleigh scattering induced by conjugation of lysozyme to the surface of gold nanoparticles, glass slides were cleaned in piranha solution (sulfuric acid: hydrogen peroxide = 7 : 3 v/v) for 60 minutes and then rinsed with ethanol. The cleaned glass slides were modified with 1 mM APTES in ethanol for 5 hours. The modified glasses were then rinsed with ethanol and followed by drying with N2 gas. In order to immobilize individual nanoparticles on the glass slides, a small aliquot (ca. 10 µL) of the diluted gold nanoparticle solution was dropped onto the APTES modified glass slide and reacted with the APTES only for 30 seconds. To remove physically adsorbed nanoparticles, the glass slide was rinsed with DI water and dried with N2 gas. Rayleigh scattering spectra for single gold nanoparticles were collected via dark-field transmission optical microscope (Olympus BX43, Tokyo, Japan) equipped with hyperspectral imaging spectrophotometer (CytoViva Hyperspectral Imaging System, Auburn, AL, USA). A 20× objective lens for 6 ACS Paragon Plus Environment

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imaging was used and integration time for collecting spectra was 0.25 seconds. Spectra were collected before and after sequential addition of 2 mM Ni ion solution with 10 µM lysozyme to the nanoparticles immobilized on the glass slides. For each sample, 30 spectra of individual nanoparticles was collected and their averaged spectra were normalized. Crystallization, data collection, and structure determination. To crystallize lysozyme, lysozyme and all gold nanoparticles were mixed in the following way: 50 µL of 470 µM lysozyme was mixed with 0.5 µL of 2.0 × 109 particles/mL nanoparticles to make 465 µM lysozyme with ca. 2.0 × 107 particles/mL gold nanoparticles, which translates to a molar ratio of lysozyme:nanoparticles = 1.4 × 1010:1. Crystals of lysozyme were grown at 20 °C for 2 weeks by the sitting-drop vapor-diffusion method, by mixing 0.5 µL of protein solution with 0.5 µL of reservoir solution. This results in about 10,000 nanoparticles per drop. MCSG-2T crystallization suite from Anatrace was used as a reservoir solution (see Supporting Information Table S1). Crystals were transferred into cryo solution composed of reservoir solutions and 20~30 % glycerol, followed by flash-freezing in liquid nitrogen. X-ray diffraction data were collected to 1.1~1.65 Å resolution at Pohang Accelerator Laboratory (PAL) beamline 5C, Korea. Data were processed with HKL2000,16 and initial models of lysozymes were obtained using the molecular replacement program of CCP4 package17 with known lysozyme structure (PDB ID: 193L) as a research model. The model was refined with REFMAC,18-19 and manual model building was performed using the COOT program.20 The Ramachandran plot produced by COOT program20 showed no outliers in the structures. The data collection and refinement statistics are summarized in Table 1. The coordinate and structure factors of lysozyme with/without nanoparticles have been deposited in the Protein Data Bank (PDB ID’s: 5K2K, 5K2N, 5K2P, 5K2Q, 5K2R, 5K2S).



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RESULTS AND DISCUSSION Conjugation of lysozyme to the surface of gold nanoparticles To accelerate the formation of nucleation cores, gold nanoparticles with different sizes and shapes (gold nanospheres: 5, 10, 20, 50, and 100 nm; gold nanostars: 15, 30, 60, 125 and 230 nm) were conjugated with hen egg-white lysozyme in the presence of Ni2+ ions. Nanoparticles were decorated with abundant carboxylic groups (-COOH), which have been shown to bind lysozyme protein by ionic interactions with Kd values between 257 µM to 417 µM.

9,21

It is also known that various amino acids such as histidine, glutamic acid, and aspartic

acid can coordinate with Ni2+ ions, which are bound by the carboxylic groups covering the surface of the gold nanoparticles.22-25 Accordingly, Ni-coordinated gold nanoparticles allow for favorable adsorption of lysozyme on their surfaces, as described in Figure 1a. To confirm the conjugation of lysozyme to the surface of the gold nanoparticles, we employed single nanoparticle dark-field spectroscopy and transmission electron microscopy (TEM). Dark-field spectroscopy has been extensively applied to characterize the molecular binding of metallic nanoparticles by measuring the Rayleigh scattering from single nanoparticles.26-30 For dark-field spectroscopy, gold nanoparticles were immobilized on a glass slide, exposed to Ni2+ ions and treated with 10 µM lysozyme. Figures 1b and 1d show representative spectral shifts after binding of lysozyme to the gold nanospheres and gold nanostars, respectively. For a single 50 nm gold nanosphere, a remarkable red-shift (ca. 16 nm) of the Rayleigh scattering band was observed after conjugation of lysozyme, which is consistent with previous reports of protein conjugation on nanoparticles.29-30 Likewise, a redshift of approximately 11 nm from the intrinsic plasmon band of individual 60 nm gold nanostars was measured after conjugation of lysozyme protein to the nanoparticles (Figure 1d). 8 ACS Paragon Plus Environment

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The TEM images in Figures 1c and 1e present direct evidences of lysozyme conjugation on the surface of the gold nanoparticles. TEM images for all sizes (i.e., 5, 10, 20, 50, and 100 nm) of gold nanospheres used in this study (Figure S1a-e) revealed that conjugation of lysozyme to the nanoparticles is size-dependent. In the case of small nanospheres, such as 5 and 10 nm, conjugation of lysozyme was hardly observed, probably owing to the small surface area for binding, as compared to the size of the lysozyme (ca. 39 Å × 39 Å × 78 Å).31-32 In cases of larger nanospheres (i.e., 20, 50, and 100 nm), thin layers, derived from the conjugation of lysozymes, were clearly observed on the smooth surfaces of the nanoparticles. Nanostars, which have rough surfaces, also showed size-dependence. In the case of 15 nm nanostars, the conjugated lysozyme was not observed on the surface of the nanoparticles owing to their small surface area for binding (Figure S1f). However, 30 nm nanostars were partially decorated with lysozyme at concave surfaces (Figure S1g). For large nanostars (i.e., 60, 125 and 230 nm nanostars; Figure S1h-j), lysozyme entirely covered the rough surfaces. These results indicate that the size and shape of gold nanoparticles used as nucleation cores can affect the efficiency of protein crystallization. Moreover, we have additionally performed conjugation of green fluorescent protein (GFP) to gold nanoparticles to visualize its conjugation by color. Fluorescence images of the single gold nanoparticle before and after treatment of GFP show the induction of bright green fluorescence by GFP around gold nanoparticle (Figure S2).



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Figure 1. Characterization of lysozyme-conjugated gold nanoparticles. (a) Ni-mediated lysozyme conjugation to the surface of gold nanoparticle. (b,c) A representative spectral shift of Rayleigh scattering and a TEM image for conjugation of lysozyme to a 50 nm gold nanosphere. (d,e) A representative spectral shift of Rayleigh scattering and a TEM image for conjugation of lysozyme to a 60 nm gold nanostar. All scale bars correspond to 20 nm.

Effects of gold nanoparticles on the crystallization of lysozyme Using lysozymes and various gold nanoparticles, we next examined the effects of gold nanoparticles on protein crystallization via the sitting-drop vapor diffusion method (see also Scheme 1), which has been extensively applied in protein crystallization.33 Figures 2a and 2b show crystallization conditions in the presence of gold nanospheres and gold nanostars, respectively. Five different gold nanospheres (diameters of 5, 10, 20, 50, and 100 nm) and five gold nanostars (15, 30, 60, 125, and 230 nm) were used to observe the size- and shape-dependent effects on lysozyme crystallization. Figure 2c shows that five replicate experiments were performed on control groups for fair comparison with the gold 10 ACS Paragon Plus Environment

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nanoparticles. Red-colored boxes in Figures 2a and 2b indicate newly observed crystallization conditions as compared with the control groups (Figure 2c). Figures 2d-f show the summation of crystallization conditions from five nanosphere (Figure 2d), five nanostar (Figure 2e), and five control groups (Figure 2f), respectively. Charts of the crystallization cases for the nanosphere group (Figure 2g) and nanostar group (Figure 2h) summarize the size-dependent tendency as compared to the control group in Figure 2c. Overall, crystallization trials with gold nanospheres and gold nanostars produced a significantly increased number of crystallization conditions than control groups (Figure 2d-f and 2i). The following are crystallization conditions observed only in the nanoparticle groups; 5 nm diameter gold nanosphere (G6), 20 nm gold nanosphere (A2, D5, and D12), 50 nm gold nanosphere (E10), 100 nm gold nanosphere (B6, D6, D12, E2, E9, and G6), 30 nm gold nanostar (A10 and D5), 60 nm gold nanostar (D1), 125 nm gold nanostar (A8 and D5), and 230 nm gold nanostar (A10, D5, E2, and E10). The number of crystallization conditions increased from 37 to 46 (24 % increase) with gold nanospheres (Figure 2d), and to 43 with gold nanostars (16 % increase; Figure 2e), respectively. Crystallization conditions observed in control groups also produced crystals with gold nanoparticles, except only one condition A11 in the case of gold nanospheres. In general, the larger the diameters of the nanoparticles, the more crystallization conditions were detected. The largest nanoparticles used in our study, 100 nm gold nanospheres and 230 nm gold nanostars, produced the highest number of new crystallization conditions (six and four conditions, respectively). This is in agreement with our observation that larger gold particles tend to bind lysozyme more efficiently, as well as with a theoretical study that found that seed particles with larger diameters are more effective in inducing nucleation.10 Figures 2d, 2e, and 2i show that the shape of gold nanospheres with smooth surface can be more effective for the crystallization of lysozyme as compared to rough nanostars. 11 ACS Paragon Plus Environment


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To further investigate whether gold nanoparticles can accelerate crystallization kinetics, we monitored the numbers of crystallization conditions in a time-lapse manner for 14 days after preparing sitting-drop crystallization plates. The rates of crystallization were compared with those of the control group, and Figure S3 shows that gold nanoparticles could increase the rate of crystallization. Gold nanospheres, in all cases, resulted in earlier crystal formation than that in control groups. Figure 3 shows representative microscopic images of lysozyme crystals formed using gold nanospheres (Figure 3a-e) or gold nanostars (Figure 3fj). Lysozyme crystals formed without gold nanoparticles (control group) are also shown in Figure S4. As another test case, same set of crystallization experiments using bovine serum albumin (BSA) were performed with gold nanospheres (diameters of 5, 10, 20, 50, and 100 nm), gold nanostars (15, 30, 60, 125, and 230 nm) and five replicate controls without gold nanoparticles (Figure S5). Numbers of observed crystallization conditions were 5, 7 and 5 in gold nanospheres, gold nanostars and control groups, respectively and total of three new crystallization conditions were found in the presence of gold nanoparticles (red boxes in Figure S5). This result shows gold nanoparticles can enhance the crystallization of various proteins. Finding new crystallization conditions using gold nanoparticles is advantageous for protein structure determination by X-ray crystallography because the quality of crystals, including diffraction limits, ease of derivatization by heavy atoms, and robustness, often depends on the crystallization condition.


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Figure 2. Crystallization of lysozyme with and without gold nanoparticles. (a,b) Crystallization conditions observed in 96-well crystallization plates of lysozyme solution with gold nanoparticles (colored blue and red). Conditions produced in both control groups and gold nanoparticle groups are in blue. Conditions produced only with gold nanoparticles are in red. (c) Crystallization conditions observed in 96-well crystallization plates of lysozyme solution without gold nanoparticles (colored green). (d-f) Summation of crystallization conditions of (a-c). In (a-f), the numbers in parentheses indicate the number of crystallization conditions under each condition. (g,h) Charts of the number of crystallization



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conditions according to the size of gold nanospheres (g) and gold nanostars (h). (i) A summary chart of the nanoparticle shape-dependent crystallization tendency.

Figure 3. Representative microscopic images of lysozyme crystals. (a-e) Images of lysozyme crystals formed in the presence of gold nanospheres: 5 nm (a), 10 nm (b), 20 nm (c), 50 nm (d), and 100 nm (e). (f-j) Images of lysozyme crystals formed in the presence of gold nanostars: 15 nm (f), 30 nm (g), 60 nm (h), 125 nm (i), and 230 nm (j). Scale bars correspond to 500 µm.

Data collection and structure determination of lysozyme To determine whether gold nanoparticles interfere with data collection and/or structure determination, multiple crystals grown with or without gold nanoparticles (denoted with asterisks in Figure 2a-f) were used for structure determination. Crystals grown in the presence of gold nanoparticles were washed five times to remove excess gold nanoparticles with reservoir solution, and then frozen in liquid nitrogen for data collection. Crystals formed with gold nanoparticles produced a weak gold (Au) signal, as detected by X-ray fluorescence spectroscopy (XFS). Six conditions (denoted with asterisks) in Figure 2a-c were selected to determine crystal structures: two pairs of crystals produced both by control and with gold 14 ACS Paragon Plus Environment

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nanoparticle conditions and two other crystals produced only with gold nanoparticles. These six structures were deposited in the protein data bank (PDB) with PDB IDs: 5K2P (controlC11), 5K2Q (20 nm gold nanostar-C11), 5K2K (control-C12), 5K2N (20 nm gold nanosphere-C12), 5K2R (30 nm gold nanostar-D1), and 5K2S (100 nm gold nanosphere-G6). Data collection and refinement statistics of these six crystals are summarized in Table 1. All the crystals belonged to space group P43212 with very similar cell dimensions. We note that there were no problems nor special procedures needed for structure determination despite the use of gold nanoparticles. We also found that when the crystallization conditions were same crystals obtained in the presence of gold nanoparticles showed better resolution than control groups (5K2Q and 5K2N had better resolution than 5K2P and 5K2K, respectively). Comparison of the final structures showed that they were very similar with each other with root-mean-square deviation (RMSD) values ranging from 0.098 to 0.252 Å, indicating that gold nanoparticles do not cause significant structural changes (Table S2 and Figure S6).



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Table 1. Data collection and refinement statistics

*Rsym =

∑|| ∑

,

where I is the intensity of an individual reflection and is the average

intensity over symmetry equivalents

CONCLUSIONS In summary, two types of gold nanoparticles (nanospheres and nanostars) of varying sizes significantly increased the number of crystallization conditions and crystallization rate of hen egg-white lysozyme as compared to control groups. In addition, we verified that use of gold nanoparticles did not have adverse effects on data collection, structure determination, or the final crystal structure. Our results suggest gold nanoparticles can increase the success rate of crystallization. Since crystallization is one of the most time-consuming and erratic steps in protein structure determination, the use of gold nanoparticles as a nucleation-inducing reagent can greatly facilitate the structure determination process of proteins.


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ASSOCIATED CONTENT Supporting Information TEM images, characterization of protein conjugation, crystallization conditions and kinetics are in the supplemental materials. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] * E-mail: [email protected] Author Contributions †

S.K. and H. Y. K. contributed equally to this work.

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (2015R1D1A1A02062259 for J. Choe and I. Choi, and 2014R1A1A1038069 for I. Choi).



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Gold Nanoparticles as Nucleation-Inducing Reagents for Protein Crystallization Sanga Ko,† Hye Young Kim,† Inhee Choi,* and Jungwoo Choe*

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Synopsis In this research, we propose the use of gold nanoparticles to enhance nucleation and crystal formation. Use of differently sized gold nanospheres and gold nanostars showed significantly increased number of crystallization conditions (by about 20 %) of hen egg-white lysozyme. Furthermore, the use of gold nanoparticles did not have any adverse effect on data collection or structure determination.


Gold Nanoparticles as Nucleation-Inducing Reagents for Protein

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