Real-Time Control of Nanoscale Protein Assembly for Further

Jul 13, 2012 - ABSTRACT: In this study, a solution circulating apparatus was employed to control the aggregation of protein molecules in situ. The ass...
0 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/crystal

Real-Time Control of Nanoscale Protein Assembly for Further Crystallization Using a Solution Circulating Nanoaggregation Control Apparatus Hideyuki Miyatake* and Naoshi Dohmae RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan S Supporting Information *

ABSTRACT: In this study, a solution circulating apparatus was employed to control the aggregation of protein molecules in situ. The association state of protein molecules was controlled by injecting protein solutions, buffers, and solutes into the device while monitoring the heterodispersity of the molecular nanoaggregates by an apparatus of high-performance heterodyne-based dynamic light scattering. Further, a highly ordered association of protein nanoaggregates was induced that lead to the generation and growth of protein crystals suitable for structural analysis by the X-ray protein crystallography. We could also dissociate protein molecules that had already formed aggregates into monoor bimodal states and reassociate them to form protein crystals, which contribute to minimizing the amount of protein required for crystallization. Our nanotechnological approach allowed us to carefully monitor and control crystal growth with a focus on the association state of the target proteins. The high-performance DLS device monitors the in situ association process of the target proteins allowing a dynamic response to modify the solution properties and leading to a deeper understanding of protein crystallization and achieving rational control of the processes.

1. INTRODUCTION The association state of protein molecules is influenced by the physicochemical conditions in solution. In X-ray protein crystallography,1 protein crystals serve to amplify the 3D structure of each protein molecule ordered in the crystal. The typical magnification by the crystals is 108× or higher, rendering it possible to analyze protein structures at the subnano level. However, traditional protein crystallization techniques such as vapor diffusion rely on the optimization of conditions obtained from the sparse matrix or incomplete factorial screens.2,3 In these methods, we can usually select the initial crystallization conditions only once crystal growth appears or after an indication of crystallization is observed by optical microscopy. This results in considerable wastage of protein samples and lower success rates of crystallization.4 Because of the trial-anderror procedure required for determination of the initial crystallization conditions, most of the prepared protein sample gets wasted, although the protein molecules consumed in the growth of the resultant crystals are small. The situation was almost the same when recently developed robotics technology was applied for high-throughput protein crystallization.5−7 However, several studies have attempted to elucidate the mechanism of protein crystallization using physicochemical methods.8−14 A systematic study on proteins in solution has suggested that the dispersity of proteins is closely associated with the probability of crystallization.15 Recently, time-resolved DLS measurements have been proposed or conducted with the © 2012 American Chemical Society

aim of performing in situ assays to determine the probability of protein crystallization.16−19 Heterodyne interferometers are marginally more precise than conventional photon correlation DLS systems for DLS because, in the former case, Doppler shifts of wavelengths between the defocused incident light beam (reference) and the sample focused scattered light beam are detected.20−22 In the latter case, however, only the scattered beam is detected. The difference results in a better S/N of the autocorrelation function allowing to infer the exponential decay time and hence the particle radius with enhanced precision. Here, we used a heterodyne-based dynamic scattering device (NanoTrac Ultra-BIO; Microtrac Inc./Nikkiso Co. Ltd.), to create a protein solution monitoring device for observing crystal growth and for measuring the physicochemical parameters related to crystallization, such as conductivity, protein concentration, solution temperature, and solution dispersity of the protein nanoaggregates in solution. We also freely changed the crystallization parameters during the measurement to investigate the time-resolved mechanism of protein crystallization. This equipment may help us to gain a deeper understanding of the mechanism of protein crystallization and lead to development of an automatic crystallization Received: May 8, 2012 Revised: July 10, 2012 Published: July 13, 2012 4466

dx.doi.org/10.1021/cg300619d | Cryst. Growth Des. 2012, 12, 4466−4472

Crystal Growth & Design

Article

Figure 1. (a) Schematic overview of protein nanoaggregation control apparatus. The fluid circuit was equipped with the following devices: syringe pumps (A, buffer A; B, buffer B; P, protein solution) for injection of solution into the circuit, a semipermeable membrane within the circuit for solution exchange, and a protein association control cell with a CCD camera for observation of crystal and precipitate formation. Further, the following devices were inserted for monitoring the solution state in the circuit: a conductivity meter, UV meter, and heterodyne DLS device. A peristaltic pump circulated the solution in the fluid circuit. A computer was used to integrate these devices. (b) Illustration of the protein association control cell. The heart-shaped cell aids in crystallization by reducing the fluid flow around the cell edges, as shown in the arrows in the schematic. CH3COOH, pH 4.3) and B (buffer A + 2.0 M NaCl) were used for equilibration and precipitation. 2.2. Constructing a Protein Nanoaggregation Control Apparatus. The overview of the current protein nanoaggregation control apparatus and the view of the protein association control cell are illustrated in Figure 1a,b, respectively. Figure 2a shows the flow adaptor set on the heterodyne-based DLS apparatus (NanoTrac UltraBio; Microtrac Inc./Nikkiso Co. Ltd.). The temperature of the nanoaggregation control cell was maintained at 293 K during all the

device that facilitates a more controlled and efficient crystallization of proteins.

2. EXPERIMENTAL SECTION 2.1. Preparation of Protein and Buffer Solutions. The protein solution was prepared (20 mg/mL: P solution) using hen egg-white lysozyme (HEWL, MW = 14313.2; Lot. No. 129K1863, Sigma) and filtered through a syringe filtration disk prior to use (0.2 μm Minisart, Sartorius Stedim Biotech). Buffers A (0.1 M CH3COONa/ 4467

dx.doi.org/10.1021/cg300619d | Cryst. Growth Des. 2012, 12, 4466−4472

Crystal Growth & Design

Article

Figure 2. Schematic illustration of the heterodyne-based DLS apparatus. (a) Detailed schematic view of the heterodyne-based DLS apparatus NanoTrac Ultra-Bio. A flow adaptor is attached over the laser probe tip, enabling flow-through measurement. (b) Schematic illustration of the optics for laser light. A laser light (Io (ωo)) injected into the optical fiber is scattered by particles in the inside cell of the flow adaptor. The scattered light (Is (ω) from the particle then returns to the detector with the incident light Io (ω) as the control reference. The heterodyne signal is measured by the detector as the difference in frequency of ωs − ωo. 2.3. DLS Measurement and Analysis. DLS analysis is a method for measuring particle sizes ranging from nanometers to micrometers. DLS measures particle motion optically in a fluid of known temperature and viscosity. The suspended particles are illuminated with a coherent light source. The light scattered from the suspended particles exhibits frequency shifts according to the time-dependent position or velocity of the suspended particles. Over time, random particle motion forms a distribution of such shifts in the optical frequency. The distribution of frequencies is representative of the particle size of the suspended particles. The power spectra for heterodyne detection are represented in the equations given below:

experiments. Figure 2b depicts the optics for the measurement. In the current study, we used lysozyme as the test protein sample. A solution circulating association control apparatus was constructed such that we could increase the concentrations of protein and solutes by monitoring the solution conditions with several sensor devices. In the apparatus, a solution circuit made of a Teflon tube (ϕout = 1.0 mm; ϕin = 0.8 mm) was inserted by the components such as a flow-through semipermeable membrane (Vivaflow 50, 5000 MWCO; Sartorius Stedim Biotech), a protein nanoaggregation control cell thermocontrolled by a water circulating chiller, a conductivity meter, an UV absorption meter, a heterodyne-based dynamic light scattering (DLS) apparatus, and solution injection ports connected with injection syringes. The fluid in the tubing is compulsively circulated by the peristaltic pump. When the protein solution is injected into the apparatus, the protein becomes concentrated according to the amount of solution injected through the semipermeable membrane. In addition, introducing concentrated precipitant solution (e.g., concentration = Co) through the semipermeable membrane can increase the concentration of the solute to the initial concentration of the precipitant (Co). The protein nanoaggregation control cell is made of transparent acrylic resin to facilitate observation using a CCD camera (Links Co. Ltd.; Basler Scout series) with a CVL0310X-MIMP lens (CBC America Corp). Temperature was controlled using Neslab Digital Plus RTE 7 RTE7 Chiller Recirculating Water Bath Circulator (Thermo Scientific). In addition, the unit contained a peristaltic pump (MINIPULS 3, Gilson; ϕ = 0.5 mm PVC tube; M&S Instruments Inc.), a UV absorption monitor (AC-5200 L, 0.2 mm absorption cell; ATTO Corp.), a conductivity sensor (conduct meter, DS52; flow-through sensor, 3574−10C; Horiba Co. Ltd.), and a heterodyne DLS device (NanoTrac ULTRA-BIO; Microtrac Inc./ Nikkiso Co. Ltd.). The total circuit volume was 3.0 mL. Throughout the course of experiment, the protein nanoaggregation controlling cell was maintained at 293 K. Further, the heterodyne-based DLS device can be used for monitoring the dispersity of the protein nanostructures over a wide range of concentrations (μM to mM), even in the flowing protein solution. One of the outstanding features of the heterodyne-based DLS is the larger dynamic range for measurement than the homodynebased DLS. The detail is described in ref 21.

P(ω) = io⟨is⟩ ωo =

2ωo ω2 + (ωo)2

1 8πkT sin 2(θ /2) r 3λ 2η

(1) (2)

The parameters determining the power spectrum P(ω) are as follows: λ = wavelength in suspending medium; ω = frequency; ωο= frequency from particle at half height; η = viscosity; θ = scatter angle; is = scattered optical intensity;, io = reference optical intensity; r = particle radius; k = Boltzmann’s constant; and T = temperature. In this study, we defined the variation in heterodispersity of the protein nanoaggregates by using a standard deviation (SD) calculated according to the equation given below:

SD = (d84 − d16)/2

(3)

where d84 is the particle diameter corresponding to 84% of the population in the accumulated population curve, and d16 is the particle diameter corresponding to 16% of the population in the accumulated population curve The power spectrum has a Lorentzian function, as shown in eq 1. The characteristic frequency ωο in eq 2 is inversely proportional to the particle size and represents the half-power point of the spectrum. Since the signal level for heterodyne power is proportional to the product of the scattered (is) and reference (io) intensities, the weak signal acquired from minute dispersion particles such as protein molecules is amplified, leading to fine S/N measurement. The detector signal is digitized, and the frequency power spectrum of the signal is 4468

dx.doi.org/10.1021/cg300619d | Cryst. Growth Des. 2012, 12, 4466−4472

Crystal Growth & Design

Article

determined using fast Fourier transform (FFT) digital signal processing. The temperature inside the protein association control cell is controlled with the water jacket. Each syringe pump pours buffer A (equilibration buffer), buffer B (precipitant solution), and P (protein solution) into the solution circuit. The peristaltic pump pumps the solution through the solution circuit. An automatic measurement program (written using LabVIEW) installed in a Windows PC was used to measure the solution state in the circuit via each measurement apparatus.

3. RESULTS AND DISCUSSION 3.1. Simultaneous Injection of Lysozyme and Precipitant Gradually from Zero Concentrations. To examine the ability of the current apparatus to arbitrarily change the concentrations of proteins and precipitants, we simultaneously injected lysozyme and precipitant into the apparatus at 0.1 and 0.2 mL/h, respectively, as shown in the phase diagram (Figure 3, line c). The absorption at 280 nm (λ280) started to increase at

Figure 3. Phase-diagram of lysozyme and precipitant concentrations. (a) Rapid injection of the precipitant to the concentrated lysozyme solution, followed by gradual injection of the precipitant. This process corresponds to the batch method for protein crystallization. (b) Rapid injection of the precipitant into the already concentrated lysozyme solution, followed by simultaneous injection of the lysozyme and the precipitant. This process corresponds to the hanging (or sitting) drop vapor diffusion technique for protein crystallization. (c) Gradual and simultaneous injection of lysozyme and precipitant.

65 (×10) min because the initial state inside of the circuit is free from proteins, time-lag for the initial detection for increasing the λ280 owing to the adsorption of the protein molecules mainly to the semipermeable membrane, even the protein and precipitant solutions are simultaneously injected into the circuit. During the simultaneous injections of solutions P and B until 172 (×10) min, we were able to maintain heterodispersity (SD < 2.0 nm). However, heterodispersity was achieved at 173 (×10) min, and the precipitant appeared in the protein nanoaggregation control cell. Thereafter, we were unable to recover the homodispersity by injecting buffer A or by reducing the conductivity (Figure 4a; Supporting

Figure 4. Results of control experiments of lysozyme nanoaggregation. The upper heat maps show time-course changes in the hydrodynamic diameter of lysozyme molecules measured by the heterodyne-based DLS apparatus. The measurements were conducted every 10 min. The middle charts represent the conductivity (bold line) and lysozyme concentration as monitored by the absorbance at 280 nm (thin line). The lower charts represent the time-course changes in dispersity (SD). The images of the protein association control cell are shown. (a) Gradual injection of lysozyme and precipitant into the circuit. The lysozyme and precipitant solutions were homodisperse at 70 (×10 min) to 170 (×10 min) but rapidly changed to the heterodisperse state. Finally, only the precipitant appeared in the control cell. (b) 4469

dx.doi.org/10.1021/cg300619d | Cryst. Growth Des. 2012, 12, 4466−4472

Crystal Growth & Design

Article

in the protein nanoaggregation control cell increased to approximately 0.5 mm (Figure 4b; Supporting Information Video S4b). These results indicate that the nanoaggregation state of lysozyme immediately improved after the rapid injection of the precipitant. The precipitant is added at a high speed when the initial droplet for vapor diffusion is prepared. The rapid injection may induce protein molecules to become supersaturated, which is considered mandatory for the protein crystallization process to occur. This process corresponds to the typical step in the vapor diffusion technique when droplets of protein solution and precipitant are mixed to form initial nucleation units. This process is schematically illustrated in Figure 5a.

Figure 4. continued

Rapid addition of the precipitant (at 79 × 10 min) to the already concentrated lysozyme solution (15 mg/mL), followed by the gradual addition of the precipitant solution. The dispersity improved (SD: 1.7 → 1.4) after rapid addition of the precipitant, and the solutions remained homodispersed until the end of the experiment. Finally, some crystals grew in the cell. (c) Rapid injection of precipitant solution into the lysozyme solution (15 mg/mL), followed by gradual injections of lysozyme (0.1 mL/h) and precipitant (0.2 mL/h). The dispersity improved after the rapid injection of precipitant, and the solution remained homodispersed throughout the gradual injection of the lysozyme and precipitant solutions. The lysozyme crystals grew in the cell. (d) Rapid injection of the precipitant into the heavily heterodisperse lysozyme solution (15 mg/mL). The dispersity did not improve by this rapid injection. Slow injection of the lysozyme and precipitant solutions improved the dispersity to homodispersed (indicated by red arrows), and crystal growth was initiated. The dispersity worsened again, and the crystals reached approximately 0.5 mm in size, and precipitation occurred. Information Video S4a). This result suggests that rapid increase of the precipitant concentration in the protein−precipitant mixtures, which usually occurs in the conventional crystallization techniques such as vapor diffusion and batch experiments, may be inevitable to maintain the dispersity of the solutions. 3.2. Simulating a Batch Experiment. 3.2.1. Initial Equilibration Step. Then, we simulated the phase-diagram that occurred in the batch technique (Figure 3, line a) by the following steps: the solution circuit in the apparatus was equilibrated with buffer A circulated by the peristaltic pump at 10 rpm (0.25 mL/min), to attain a conductivity of 0.5 S/m. Then, lysozyme solution (P solution) was injected into the circuit at a concentration of 20 mg/mL (the absorbance intensity at λ280 = ∼1.0) while rotating the peristaltic pump at 5 rpm into the circuit (0.125 mL/min) to attain constant concentrations. 3.2.2. Rapid Injection of Precipitant into Lysozyme Solution. Lastly, 1.3 mL of precipitant (buffer B) was rapidly (1.0 mL/min) injected into the solution circuit while intensively rotating the peristaltic pump at 50 rpm (1.25 mL/ min) for 10 min. 3.2.3. Gradual Injection of Precipitant into Lysozyme Solution. After the rapid precipitant injection, the peristaltic pump speed was reduced to 0.2 rpm (0.005 mL/min), and then the precipitant (Buffer B) was gradually (0.1 mL/hour) injected into the circuit. The aggregation state of the lysozyme molecules and other solution parameters were measured every 10 min. While taking the measurement, the peristaltic pump speed was reduced to 0.1 rpm (0.0025 mL/min). After taking the measurements, the peristaltic pump speed was again increased to 0.2 rpm (0.005 mL/min). The conductivity of the solution increased from approximately 0.6 S/m to 4.5 S/m, which corresponds to 2 M × 4.5/10 = 0.9 M NaCl, where the conductivity of the precipitant solution (buffer B: 2.0 M NaCl in buffer A) was 10 S/m. After the rapid mixing of buffer B, the dispersity immediately changed from 1.7 to 1.4 nm, maintaining the homodispersity (SD < 2.0 nm) during the gradual (0.1 mL/ h) injection of buffer B. Finally, the size of the lysozyme crystals

Figure 5. Schematic illustrations of the time-course association/ dissociation behavior of lysozyme molecules in the device. (a) The rapid addition of precipitant to protein solution can improve the dispersity to the homodispersed state. Gradual injection of the precipitant thereafter induces protein molecules to form crystals. (b) Gradual addition of the lysozyme and precipitant solutions to the heavily aggregated protein solution can improve the heteroaggregation state to homodispersed and promote subsequent crystallization.

3.3. Simulating a Vapor-Diffusion Experiment. 3.3.1. Initial Equilibration Step. We simulated the phase transition that occurs in the hanging (or sitting) drop vapor diffusion technique for protein crystallization (Figure 3, line b). The solution circuit was equilibrated with buffer A, and then the lysozyme solution (solution P) was injected at the concentration of 15 mg/mL (the absorbance intensity at λ280 = ∼0.75). 3.3.2. Rapid Injection of Precipitant Solution Followed by Gradual Injection of Lysozyme and Precipitant Solutions. After the rapid injection of 1.3 mL precipitant (buffer B), the dispersity of the lysozyme was improved from a high heterodispersity (SD = ∼60 nm) to a good homodispersity (SD = ∼1 nm) (Figure 4c; Supporting Information Video S4c). Then, the lysozyme (solution P) and precipitant (buffer B) were gradually (0.1 mL/h and 0.2 mL/h, respectively) injected into the circuit. The homodispersity was kept up to 230 (×10) min, after which the dispersity rapidly became heterodispersed. We could grow large lysozyme crystals (0.5 mm) using this procedure. 3.3.3. Validating Quality of the Lysozyme Crystals. We carried out an X-ray diffraction experiment23 to validate the 4470

dx.doi.org/10.1021/cg300619d | Cryst. Growth Des. 2012, 12, 4466−4472

Crystal Growth & Design

Article

showing details of the X-ray crystallographic experiments. This material is available free of charge via the Internet at http:// pubs.acs.org.

quality of the prepared crystals. We could collect X-ray diffraction data at ∼1.0 Å resolution24 and then solve the crystal structure (Supporting Information Figure S1, Table S1).25 The obtained data quality of resolution ranks third among the 45 X-ray structures of HEW lysozyme deposited in the current PDB. This result suggests the outstanding potential of the present nanotechnological approach to control protein nanoaggregation, which intensively correlates with the quality of crystal growth that follows. In the current study, we did not have a chance to collect the X-ray data of the crystals prepared from the heterodispersed solution. This result shows that we can prepare high-quality single crystals of proteins by the apparatus controlling the solution state. 3.4. Converting Heterodisperse Lysozyme Solution to the Homodisperse State. We also aimed to improve the dispersity of the heterodisperse lysozyme solution prepared by equilibrating the lysozyme precipitant. We rapidly injected the precipitant (buffer B) into the heterodisperse (SD > 1.0E03 nm) lysozyme solution (15 mg/mL), but this did not result in any changes in the dispersity. Then, we gradually injected the lysozyme solution (solution P) (0.1 mL/h) and the precipitant solution (buffer B) (0.2 mL/h) into the circuit, which corresponds to the transition in the phase-diagram during the vapor diffusion. Then, the dispersity of the protein became bimodal (2 major peaks, SD < 10) at 90 (×10) min, and the crystals started to grow (Figure 4d; Supporting Information Video S4d). Until 230 (×10) min, the bimodal state continued and the crystals were growing. In this procedure, although the initial dispersity of the lysozyme nanoaggregation in solution appeared unsuitable for crystallization, we successfully controlled the dispersity, rendering the molecules suitable for crystallization. This result suggests that even lysozyme aggregates can be dissociated by simultaneous addition of lysozyme and precipitant. In the current apparatus, it was necessary to induce forced circulation of the protein and the buffer solutions by the peristaltic pump in order to ensure homogeneous injections. Some researchers have debated whether active stirring of the solution during crystallization is effective,26−30 although most researchers agree that suitable conditions will ensure good crystallization, regardless of conditions such as agitation and gravity.31,32 In this study, we proved that peristaltic pump driven circulation in the apparatus did not inhibit crystallization. Indeed, we could prepare highquality lysozyme crystals, which diffracted X-rays to a resolution of 1.0 Å. The present apparatus allows us to lead to timeresolved dynamic control of the solution state during crystallization.16 This process is schematically illustrated in Figure 5b.

■

*Phone: +81-48-467-9510. Fax: +81-48-462-4704. E-mail: [email protected]. Author Contributions

H.M. conceived the whole project. H.M. constructed the apparatus, conducted the experiments, and wrote the entire manuscript. H.M. carried out the X-ray structural study. N.D. read the manuscript. H.M. and N.D. discussed the experimental procedures and obtained results and interpreted them together. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was financially supported by the RIKEN Collaboration Centers Program, the RIKEN Incentive Research Grant, and JSPS KAKENHI Grant Number 24651164 (to H.M.). This work was carried out with the assistance of Mrs. S. Onda and M. Ohata (Nikkiso Co. Ltd., Japan). We would like to thank Dr. P. J. Freud (Microtrac Inc., USA) for discussing the heterodyne DLS measurements. We also thank Mrs. H. Nogami, A. Nukanobu, and Y. Okada (Nikkiso Co. Ltd., Japan) for their continuous support for this study. We thank Drs. Y. Nakamura, G. Ueno, and M. Yamamoto for collecting the X-ray diffraction data in SPring-8 using the mail-in data collection system. The heterodyne-based DLS has been patented by M. N. Trainer, W. L. Wilcock, and B. M. Ence (March 1992), as “Method and Apparatus for Measuring Small Particle Size Distributions, US Patent No. 5,094,532.”

■

REFERENCES

(1) Berman, H. M.; Battistuz, T.; Bhat, T. N.; Bluhm, W. F.; Bourne, P. E.; Burkhardt, K.; Feng, Z.; Gilliland, G. L.; Iype, L.; Jain, S.; Fagan, P.; Marvin, J.; Padilla, D.; Ravichandran, V.; Schneider, B.; Thanki, N.; Weissig, H.; Westbrook, J. D.; Zardecki, C. The protein data bank. Nucleic Acids Res. 2000, 28, 235−242. (2) Carter, C. W., Jr.; Baldwin, E. T.; Frick, L. Statistical design of experiments for protein crystal growth and the use of a precrystallization assay. J. Cryst. Growth 1985, 90, 60−73. (3) Jancarik, J.; Kim, S. H. Sparse matrix sampling: a screening method for crystallization of proteins. J. Appl. Crystallogr. 1991, 24, 409−411. (4) McPherson, A. Crystallization of Biological Macromolecules. Cold Spring Harbor Press: Cold Spring Harbor, NY, 1999. (5) Stevens, R. C. High-throughput protein crystallization. Curr. Opin. Struct. Biol. 2000, 10, 558−563. (6) Hui, R.; Edwards, A. High-throughput protein crystallization. J. Struct. Biol. 2003, 142, 154−161. (7) Miyatake, H.; Kim, S. H.; Motegi, I.; Matsuzaki, H.; Kitahara, H.; Higuchi, A.; Miki, K. Development of a fully automated macromolecular crystallization/observation robotic system, HTS-80. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2005, 61, 658−663. (8) Mikol, V.; Hirsch, E.; Giegé, R. Monitoring protein crystallization by dynamic light scattering. FEBS Lett. 1989, 258, 63−66. (9) Zucker, F. H.; Zucker, F. H.; Stewart, C.; Rosa, J. D.; Kim, J.; Zhang, L.; Xiao, L.; Ross, J.; Napuli., A. J.; Mueller, N.; Castaneda, L. J.; Nakazawa Hewitt, S. R.; Arakaki, T. L.; Larson, E. T.; Subramanian, E.; Verlinde, C. L.; Fan, E.; Buckner, F. S.; Van Voorhis, W. C.; Merritt, E. A.; Hol, W. G. Prediction of protein crystallization outcome using a hybrid method. J. Struct. Biol. 2010, 171, 64−73.

4. CONCLUSIONS We monitored the changes in the association of lysozyme molecules at the nano level. We were further able to control molecule aggregation for crystal formation. Understanding protein crystallization as the process of molecular association occurring at the nanolevel will aid in using nanotechnology to control the process of protein crystallization. The device used in our study will help us to develop an automatic protein crystallization device in the future.

■

AUTHOR INFORMATION

Corresponding Author

ASSOCIATED CONTENT

S Supporting Information *

Video files that demonstrate how the apparatus works, crystallographic information files (CIFs), and figure and table 4471

dx.doi.org/10.1021/cg300619d | Cryst. Growth Des. 2012, 12, 4466−4472

Crystal Growth & Design

Article

(10) Wilson, W. W. Light scattering as a diagnostic for protein crystal growth: A practical approach. J. Struct. Biol. 2003, 142, 56−65. (11) Boyer, M.; Roy, M. O.; Jullien, M. Dynamic light scattering study of precrystallizing ribonuclease solutions. J. Cryst. Growth 1996, 167, 212−220. (12) Kadima, W.; McPherson, A.; Dunn, M. F.; Jurnak, F. A. Characterization of precrystallization aggregation of canavalin by dynamic light scattering. Biophys. J. 1990, 57, 125−132. (13) Cardinaux, F.; Zaccarelli, E.; Stradner, A.; Bucciarelli, S.; Farago, B.; Egelhaaf, S. U.; Sciortino, F.; Schurtenberger, P. Cluster-driven dynamical arrest in concentrated lysozyme solutions. J. Phys. Chem. B 2011, 115 (22), 72277−7237. (14) Stressts, A. M.; Quake, S. R. Ostwald ripening of clusters during protein crystallization. Phys. Rev. Lett. 2010, 104, 178102−178104. (15) D’Arcy, A. Crystallizing proteins: A rational approach? Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 469−471. (16) Wilson, W. W. Monitoring crystallization experiments using dynamic light scattering: Assaying and monitoring protein crystallization in solution. Methods 1990, 1, 110−117. (17) Dierks, K.; Meyer, A.; Einspahr, H.; Betzel, C. Dynamic light scattering in protein crystallization droplets: Adaptations for analysis and optimization of crystallization process. Cryst. Growth Des. 2008, 8, 1628−1634. (18) Oberthuer, D.; Melero- García, E.; Dierks, K.; Meyer, A.; Betzel, C.; Garcia-Cabsallero, A.; Gavira, J. A. Monitoring and scoring counter-diffusion protein crystallization experiments in capillaries by in situ dynamic light scattering. PLoS ONE 2012, 7 (6), e33545. (19) Ansari, R. R.; Suh, K.; Arabshahi, A.; Wilson, W. W.; Bray, T. L.; DeLucas, L. J. A fiber optic probe for monitoring protein aggregation, nucleation and crystallization. J. Cryst. Growth 1996, 168, 216−226. (20) Garcia-Caballero, A.; Gavira, J. A.; Pineda-Molina, E.; Chayen, N. E.; Govada, L.; Khurshid, S.; Saridakis, E.; Boudjemline, A.; Swann, M. J.; Stewart, P. S.; Briggs, R. A.; Kolek, S. A.; Oberthuer, D.; Dierks, K.; Betzel, C.; Santana, M.; Hobbs, J. R.; Thaw, P.; Savill, T. J.; Mesters, J. R.; Rolf Hilgenfeld, O.; Bonander, N.; Bill, R. M.; House, D.; Garston, E.; Berkshire, R. Optimization of protein crystallization: The OptiCryst project. Cryst. Growth Des. 2011, 11, 2112−2121. (21) Trainer, M. N.; Freud, P. J.; Leonard, E. M. High concentration submicron particle size distribution by dynamic light scattering. Am. Lab. 1992, 24, 34−38. (22) Dubin, S. B.; Lunacek, J. H.; Benedek, G. B. Observation of the spectrum of light scattered by solutions of biological macromolecules. Proc. Natl. Acad. Sci. U.S.A. 1967, 57, 1164−1171. (23) Okazaki, N.; Hasegawa, K.; Ueno, G.; Murakami, H.; Kumasaka, T.; Yamamoto, M. Mail-in data collection at SPring-8 protein crystallography beamlines. J. Synchrotron Radiat. 2008, 15, 288−291. (24) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. Macromol. Crystallogr., Part A 1997, 276, 307−326. (25) Adams, P. D.; Afonine, P. V.; Bunkóczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral., G. J.; GrosseKunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. 2010, D66, 213−221. (26) Nomura, H.; Katayama, K. Development of heterodyne detection of dynamic light scattering enhanced by the Talbot effect for the size measurement of nanoparticles. Anal. Sci. 2008, 24, 459− 462. (27) Adachi, H.; Takano, K.; Matsumura, H.; Inoue, T.; Mori, Y.; Sasaki, T. Protein crystal growth with a two-liquid system and stirring solution. J. Synchrotron Rad. 2004, 11, 121−124. (28) Adachi, H.; Watanabe, T.; Yoshimura, M.; Mori, Y.; Sasaki, T. Growth of protein crystal at interface between two liquids using slow cooling method. Jpn. J. Appl. Phys. 2002, 41, L726−L728. (29) Shimizu, N.; Sugiyama, S.; Maruyama, M.; Takahashi, Y.; Adachi, M.; Tamada, T.; Hidaka, K.; Hayashi, Y.; Kimura, T.; Kiso, Y.; Adachi, H.; Takano, K.; Murakami, S.; Inoue, T.; Kuroki, R.; Mori, Y.; Matsumura, H. Crystal growth procedure of HIV-1 protease-inhibitor

KNI-272 complex for neutron structural analysis at 1.9 Å resolution. Cryst. Growth Des. 2010, 10, 2990−2994. (30) Adachi, H.; Takano, K.; Yoshimura, M.; Mori, Y.; Sasaki, T. Promotion of large protein crystal growth with stirring solution. Jpn. J. Appl. Phys. 2002, 41, L1025−L1027. (31) McPherson, A.; Malkin, A. J.; Kuznetsov, Y. G.; Koszelak, S.; Wells, M.; Jenkins, G.; Howard, J.; Lawson, G. The effects of microgravity on protein crystallization: evidence for concentration gradients around growing crystals. J. Cryst Growth. 1999, 196, 572− 586. (32) DeLucas, L. J.; Smith, C. D.; Smith, H. W.; Vijay-Kumar, S.; Senadhi, S. E.; Ealick, S. E.; Carter, D. C.; Snyder, R. S.; Weber, P. C.; Salemme, F. R.; et al. Protein crystal growth in microgravity. Science 1989, 246, 651−654.

4472

dx.doi.org/10.1021/cg300619d | Cryst. Growth Des. 2012, 12, 4466−4472