Article pubs.acs.org/crystal
Effect of Audible Sound on Protein Crystallization Chen-Yan Zhang,† Yan Wang,† Robin Schubert,‡ Yue Liu,† Meng-Yin Wang,† Da Chen,† Yun-Zhu Guo,† Chen Dong,† Hui-Meng Lu,† Yong-Ming Liu,† Zi-Qing Wu,† Christian Betzel,*,‡ and Da-Chuan Yin*,† †
Institute for Special Environmental Biophysics, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, Shaanxi PR China ‡ University of Hamburg, Laboratory for Structural Biology of Infection & Inflammation, Institute for Biochemistry and Molecular Biology, Notkestrasse 85, 22607 Hamburg, Germany S Supporting Information *
ABSTRACT: The successful crystallization of proteins is important because their molecular three-dimensional structures can be obtained through X-ray diffraction when in their crystalline form. Investigation of the crystallization process is beneficial for this purpose. We have reported that protein crystallization is sensitive to audible sound, which is commonly present but is often ignored. Here we investigate the effect of audible sound parameters, especially frequency, on a protein crystallization. We show a significant facilitation of protein crystallization using 5000 Hz audible sound, possible mechanism was also tried to be clarified. Suitably controlled audible sound can be beneficial for promoting protein crystallization. Therefore, audible sound can be used as a simple tool to promote protein crystallization. In addition, the processing of other types of materials may also be affected by audible sound.
1. INTRODUCTION
Ultrasound with frequencies ranging between 20 000 and 100 000 Hz provides a similar physical environment to audible sounds (the frequency range is between 20 and 20 000 Hz), and ultrasound has been studied in the crystallization of matter. Utilization of ultrasound in crystallization dates back to 1927 when Richards and Loomis reported the effects of ultrasound on supersaturated solutions or supercooled melts.34 During the 1950s and 1960s many aspects of ultrasound were found to be useful in crystallization; consequently “sonocrystallization” as a research direction was rapidly developed.35 Ultrasound has also been used in protein crystallization. It has since been used to increase nucleation likelihood28−31 and to improve crystal quality.30 It has also been used to grow protein crystals in levitated droplets.36−38 Despite its long history and extensive investigation into the effects of ultrasound on the crystallization of matter, there are rarely reports on audible sound and its effect on protein crystallization. Practical protein crystallization is conducted in an environment full of various audible sounds, which are often ignored by researchers. Here, we investigate the effects of the frequency and amplitude of audible sound on protein crystallization and also suggest a possible mechanism. We propose that suitably controlling the sound environment can be beneficial for promoting protein crystallization.
Protein is the material basis of life, and it is the main actor in biological activity. Protein functional studies are some of most important topics in the life sciences. Information about the molecular structure of a protein is very useful for understanding a protein’s function, and the structural information can also be used to support rational drug discovery. Currently, more than 85% of protein structures are determined by X-ray diffraction analysis, and it is the main method for structure determination. The crystal is the foundation and basis for X-ray diffraction analysis, but it is often challenging to screen protein crystals.1−5 Starting from a purified soluble protein, the overall success rate of obtaining protein crystals is approximately 30%, among which only approximately 50% can yield sufficient diffraction data for structural analysis. In other words, only approximately 15% of all purified soluble proteins yield diffracting crystals. Therefore, protein crystallization is often a “bottleneck” step for structural determination using X-ray crystallography.6−9 Many studies have sought to increase the chance of obtaining X-ray diffracting crystals. It has been reported that protein crystallization is sensitive to the surrounding environment (including the physical and chemical environment), including factors, such as magnetic10−14 and electric fields,15,16 microgravity,17,18 temperature,19,20 gels,21,22 heterogeneous nucleants,23−25 light irradiation,26,27 ultrasound,28−31 stirring,32 and mechanical vibration.33 Suitable control of these environmental factors was beneficial for promoting protein crystallization. However, researchers often ignore audible sounds that are present during the crystallization process. © XXXX American Chemical Society
Received: September 1, 2015 Revised: December 15, 2015
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in the two chambers were calibrated to the same value using the temperature sensor. Software Multi-Instrument 3.2 (Virtins Technology, Singapore) was used to control the amplitude and frequencies of the audible sound. The frequency of the loudspeaker could be set in the range of 20− 20 000 Hz. The audible sound from the loudspeaker was recorded by a record pen and analyzed by Cool Edit Pro 2.0 to calibrate the frequency of the audible sound in the chamber and confirmed to be the same as that of Multi-Instrument 3.2. The ultrasound source was also used in to test the effects of sound on crystallization. The ultrasound source was a 10 W ultrasound generator that provides continuous ultrasound with periodically varying frequencies from 20 000 to 55 000 Hz in 80 s intervals (Huangmao, Shanghai, China). This generator could be installed in place of the loudspeaker inside the chamber, so that the same experimental setup could be used. 2.3. Crystallization Experiments. Two series of experiments were conducted to study the effects of audible sound on protein crystallization: (1) a crystallization reproducibility study and (2) a crystallization screening study. The sitting drop method was utilized for both experiments. All buffers used for crystallization were filtered with a 0.22 μm membrane (Millipore, Billerica, USA) before the crystallization experiment. Proteins were dissolved in their corresponding buffers (as in Table S1) and subsequently centrifuged at 12 000 rpm for 15 min. 2.3.1. Crystallization Reproducibility Study. Protein crystallization often suffers from bad reproducibility. For example, if a crystallization droplet yields a crystal, it does not mean that one can easily obtain crystals in the next droplet even though identical crystallization conditions are used. It may be difficult to reproduce crystallization results using only a limited number of crystallization droplets, and the reproducibility is increased by repeatedly doing the same experiment using a large number of droplets. Thus, through repeated efforts the crystallization success rate can be determined. The crystallization success rate is defined as the ratio of the number of crystallization droplets that yielded crystals to the total number of droplets of the same crystallization condition. If audible sound affects protein crystallization, the crystallization success rate will be affected by using audible sound in the reproducibility study. In other words, the reproducibility study can be used to confirm the effect of audible sound on protein crystallization. In this study, we used lysozyme as a model protein in the crystallization reproducibility tests, with and without audible sound irradiation. Two series of crystallization experiments were carried out: (1) crystallization of lysozyme under various audible sound frequencies (5000, 7500, 10 000, 12 000, 15 000, and 17 500 Hz) was performed with two final concentrations of lysozyme and NaCl solutions and (2) crystallization of lysozyme in audible sound (frequency 1000 Hz) at various concentrations was performed, using final concentrations of mixed lysozyme (10 and 20 mg/mL) and NaCl solutions (10, 20, 30, and 40 mg/mL). Using a crystallization robot (Screenmaker 96 + 8, Innovadyne Technologies Inc., California, USA), each of the solutions in the concentrations listed above was dispensed into two 96-well Intelli-Plate crystallization plates (Hampton Research, California, USA) to obtain 2 × 96 identical crystallization droplets. The volume of each droplet was 2 μL (1 μL protein +1 μL precipitant); the volume of the reservoir solution was 80 μL. After the crystallization trials were set up, Crystal Clear Sealing Tape (Hampton Research, California, USA) was used to seal the crystallization plates. The crystallization plates were then placed into the temperature controlled chambers for incubation, with or without sound irradiation. The plates were incubated at 293 K for 48 h. After incubation, an automated crystal image reader (XtalFinder XtalQuest Inc., Beijing, China) was used to detect the number of droplets that had produced crystals. Thus, the crystallization success rate was obtained. High-resolution images were obtained when necessary with a polarization microscope (Olympus SZX2-ILLB, Shanghai, China) that was equipped with a digital camera (Cannon EOS 550D, Beijing, China). 2.3.2. Crystallization Screening Study. Protein crystallization screening is used to identify chemical reagents that can crystallize protein. This method tests the probability of crystallization by trial and
2. EXPERIMENTAL SECTION 2.1. Materials. Fifteen commercially available proteins (in Northwestern Polytechnical University) and eight commercially available proteins (in University of Hamburg) were utilized directly without further purification (detailed information is listed in Table S1). Chemicals for buffer formulations: sodium chloride was purchased from Chemical Reagent Co. Ltd. (Shanghai, China) and Carl Roth GmbH + Co. KG (Karlsruhe, Germany); HEPES sodium was obtained from Amresco (Solon, USA) and AppliChem GmbH (Darmstadt, Germany); Sodium acetate and ammonia were obtained from the Beijing Chemical Factory (Beijing, China). Chemicals for the Index crystallization screening kit were from Hampton Research (Aliso Viejo, CA, USA). The Classics and JCSG kits were from Qiagen (Valencia, CA, USA). 2.2. Experimental Setup. Figure 1 shows the experimental configuration for the studies. The experimental setup was designed to
Figure 1. Schematic illustration of experimental configuration for studying the effect of audible sound on protein crystallization: 1, bath water circulator; 2, temperature controlled chamber; 3, crystallization plate; 4, loudspeaker; 5, computer. Audible sound was produced by the loudspeaker which was controlled by the computer. The temperature controlled chamber was connected with the bath water circulator. The chamber was well-wrapped using sound insulation materials (sound insulation cotton and deadening felt). Crystallization was carried out inside of chamber. In the control set, all the conditions were same as those described above, except that there was no loudspeaker installed in the temperature controlled chamber.
simultaneously compare protein crystallization in two environments (with and without audible sound irradiation). Temperature-controlled chambers for both the experiment and control groups were wellwrapped and insulated using sound wave absorption materials (sound insulation cotton (Owens Corning, Shanghai, China) and deadening felt (Sanchez Goma G, Guangzhou, China)) to minimize the audible sound penetration from the ambient environment. A record pen (Meizu, Zhuhai, China) was used to record the sound in both the experiment and the control chambers. Cool Edit Pro 2.0 (Adobe Systems, California, USA) was used to analyze the recorded sound and confirm that inside the well-isolated chambers of both the control and the experiment groups there were no detectable sounds coming from outside. One of the temperature-controlled chambers was installed with a loudspeaker (R19U, 2W, Edifier, Beijing, China), which was connected to a computer that could play different sounds. The temperatures of both chambers, with copper layers attached inside the chamber to enhance the heat transfer, could be simultaneously controlled to the same temperature within a difference of 0.1 K by using a programmable water bath circulator (Polyscience 9712 refrigerated circulator, Polyscience Inc., Pennsylvania, USA). The inner size of the temperature controllers was 28 cm × 18 cm × 13 cm. The loudspeaker was fixed directly above the sample position, where the crystallization plate was placed in the temperature-controlled chamber. The distance between the loudspeaker and the crystallization plate was 7.8 cm. Before the start of the experiment, the temperatures B
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Figure 2. Results of crystallization reproducibility study with and without audible sounds. (a) Effect of audible sound on crystallization reproducibility of lysozyme at different SILs. (Note: The maximum output level of the machine was defined as 0 dB, so the sound intensity level here was relative value.) Lysozyme and NaCl concentration (after mixing) was 20 and 20 mg/mL, respectively. Frequency of audible sound was set 1000 Hz. (b) Ratio of crystallization success rate with audible sound to that without audible sound at different frequencies. Lysozyme concentrations (after mixing) were 20 and 15 mg/mL, and NaCl concentration was 30 mg/mL. An SIL of −5 dB was used to study the effect of varying frequency on protein crystallization. (c) Crystallization success rate at 10 mg/mL lysozyme (after mixing). (d) Crystallization success rate at 20 mg/mL lysozyme (after mixing). In panels a, c, and d, sound frequency was 1000 Hz. The sound intensity level was set to −5 dB in panels c and d (Error bar = standard deviation; n = 4). error, and many different combinations of chemical reagents are mixed with the protein solution. In a standard crystallization screening experiment, the protein solution is typically mixed with a certain number (e.g., 96) of crystallization reagents. The mixed solutions are incubated at a specified temperature for a period of time. After the incubation, the conditions that successfully yield crystals are identified as “hits”. In this study, two series of experiments were carried out. First, crystallization screening under different sound frequencies was performed using lysozyme and concanavalin A as the model proteins. The initial concentration of proteins was 20 mg/mL, and the sound frequencies tested were 500, 1000, 5000, 7500, 10 000, 12 000, 15 000, and 175 000 Hz. Second, crystallization screening was performed for 15 different commercially available proteins under the audible sound frequencies 1000 and 5000 Hz. The initial concentrations were 7 mg/ mL for glucose isomerase, 21 mg/mL for papain, and 20 mg/mL for the remaining 13 proteins. The information on these proteins is listed in Table S1. Crystallization screening experiments were conducted following a standard sitting drop screening protocol. The crystallization trials were set up in 96-well crystallization plates by mixing 1 μL of crystallization reagents from the screening kit Index (Hampton research) with 1 μL of protein solution using a crystallization robot. Then, the crystallization plates were placed in temperature controlled chambers for incubation at 293 K for 48 h. After incubation, the crystallization droplets were examined using an automated crystal image reader to identify the conditions that yielded crystals (referred to as “hits”). 2.4. Verification of the Effects of Sound on Protein Crystallization at the University of Hamburg. To confirm the experimental results found at Northwestern Polytechnical University, a series of verification studies were carried out at the University of Hamburg. This included two types of experiments: (1) a crystallization screening study and (2) a dynamic light scattering (DLS) study of the crystallization process. 2.4.1. Crystallization Screening Study. A second set of crystallization setups were manufactured in Germany according to
the above descriptions, but using the 5 cm thick sound wave absorption material Basotect Schaumstof f (Flexolan, Diedorf, Germany) and a different loudspeaker SoundDisc (Cabstone, Braunschweig, Germany). This setup was used to verify the screening experiments using eight different proteins (detailed information is listed in Table S1). In total, 21 comparisons (refer to Table S2 for detailed information) were conducted. In each comparison, a crystallization screen of a protein was carried out with and without sound irradiation (5000 Hz). 2.4.2. DLS Study of the Crystallization Process. To further confirm that sound irradiation affected the crystallization process, we used a XtalController900 (XtalConcepts, Hamburg, Germany) to monitor particle size distributions in solution during the crystallization process with and without sound irradiation.39 The sound frequency was 5000 Hz and 30 mg/mL lysozyme was used for the size distribution test. After starting the experiment, an equal volume of NaCl (60 mg/mL) was added (4.7 μL + 4.7 μL) to the protein solution. The initial hydrodynamic radius of monomeric lysozyme as determined by DLS was approximately 1.5 nm. The measurement lasted 40 s and was repeated every 5 min during the experiment. To minimize evaporation, the humidity was maintained near saturation. Remaining evaporation of water was detected by an ultra micro balance and was counterbalanced by injecting ∼70 pl water droplets into the crystallization drop. After approximately 24 h, the volume of the crystallization drop was reduced by 20% and subsequently kept constant for 30 h. 2.5. Solvent Evaporation with and without Audible Sound Irradiation. Audible sound may affect the evaporation of solvent in crystallization droplets and consequently influence the crystallization. We conducted an experiment to compare solvent evaporation with and without sound irradiation. Each crystallization droplet was prepared in the cap of a 0.5 mL Eppendorf tube by mixing 2 μL of 30 mg/mL lysozyme and 2 μL of 100 mg/mL NaCl. The buffer was an aqueous solution of sodium acetate at pH 4.6. The droplet on the Eppendorf tube cap was then sealed in a bottle (inner dimension height = 5 cm, C
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Figure 3. Crystallization screening results with and without audible sounds. (a) Ratio of crystallization screening hits with audible sound irradiation to that without sound irradiation. The initial concentrations for lysozyme and concanavalin A were both 10 mg/mL. (Error bar: standard deviation, n = 4). (b) Crystallization hits at 1000 Hz. (error bar = standard deviation, n = 4). (c) Crystallization hits at 5000 Hz. (error bar = standard deviation, n = 4). (d) Number of crystallization hits at 1000 (red circles) and 5000 Hz (blue triangle) normalized to the number of hits without sound irradiation. Note that in panels b and c, the lot numbers for proteinase K are different (see Supporting Table S1). diameter = 1.5 cm), and the bottle was placed in one of the temperature-controlled chambers for incubation at 293 K for 12, 24, 36, and 48 h. Each crystallization experiment was repeated ten times for a given incubation period. After incubation, the amount of evaporated water was determined by weighing the droplets using a high precision microbalance (Sartorius BS124S, Gottingen, Germany). The final results were analyzed using SPSS version 16.0 (IBM, New York, USA).
success rate for both concentration levels, showing that audible sound frequencies had a nonlinear effect on lysozyme crystallization. The mechanisms for this phenomenon may be related to many factors that will be considered in the Discussion section. Another mentionable phenomenon was that the effect of audible sound on crystallization was concentration-dependent. At the tested concentrations, the increase in the crystallization success rate was more marked when the concentration of lysozyme was 15 mg/mL than at 20 mg/mL (Figure 2b). Using the data in Figure 2b, we calculated the average ratio of crystallization success rate found under audible sound to that in the absence of audible sound. The average ratio was approximately 2.9, showing that the crystallization success rate in the presence of audible sound was approximately 1.9 times higher than that without sound. By using one-sample ttest, the ratio was found to be in the range 1.47−6.67 with 95% confidence (P = 0.0001 < 0.05), showing that the increase in the crystallization success rate when using audible sound irradiation was significant. The above results indicate that the crystallization success rate was concentration-dependent. We obtained more information by carrying out a crystallization reproducibility study with and without sound irradiation at different salt and protein concentrations. As examples of low and high protein concentrations, Lysozyme concentrations of 10 and 20 mg/ mL were selected for further investigation. The frequency of the sound was arbitrarily set to 1000 Hz. Figures 2c and 2d show that the crystallization success rate was concentrationdependent. At a very low concentration (Figure 2c, 10 mg/mL lysozyme and 10 mg/mL NaCl), the crystallization success rate was 0, with or without sound irradiation. At high concentration (Figure 2d, 20 mg/mL lysozyme and 40 mg/mL NaCl), the crystallization success rate was 100% under both sound
3. RESULTS 3.1. Effect of Audible Sound on Protein Crystallization. Sound intensity level (SIL) and frequency are important parameters of audible sound. We performed a crystallization reproducibility study by varying these two parameters to determine the crystallization success rate of lysozyme with and without audible sound. In studying the effects of the sound intensity level on crystallization, we arbitrarily set the frequency of the audible sound at 1000 Hz. The maximum output level of the machine was defined as 0 dB, so the sound intensity levels used here (−5, −12, −18, −27, and −35 dB) are relative values. Figure 2a shows the effects of the SIL on the crystallization success rate in the reproducibility study. The results indicate that the crystallization success rate increased with SILs (Figure 2a). According to these results, we subsequently used an SIL of −5 dB for studying the effect of varying frequency on protein crystallization. Figure 2b compares the ratio of crystallization success rate in cases with audible sound at different frequencies to that without sound. The frequencies tested ranged from 500 to 17500 Hz. In the tested range, the crystallization success rate of lysozyme was frequency-dependent. At very low frequencies (e.g., 500 Hz) and very high frequencies (e.g., 17 500 Hz), the crystallization success rate was increased but not as dramatically as at intermediate frequencies. At a frequency of approximately 5000 Hz, a maximum increase was observed in the crystallization D
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Figure 4. Solvent evaporation, solution temperature, and particle size in crystallization droplets under sound irradiation. (a) Amount of evaporated water over time (error bar = standard deviation, n = 10). (b) Temperature evolution of deionized water. (c) Box plot with quartiles (upper values 75%, median values 50%, and lower values 25%) of the radius distribution recorded by the XtalController 900 with and without 5000 Hz sound irradiation. Lysozyme and NaCl concentrations were 30 and 60 mg/mL, respectively (n = 3). Particles with a radius between 100 and 10000 nm were taken into account. Outliers with a score greater than 1.5 times the inner quartile range (IQR) and extreme values are excluded. IQR for the control measurement without sound was 2895 nm, whereas the IQR for the sound irradiation was much smaller (495 nm).
screening hits, we normalized the hits based on the hits without sound irradiation. After normalization, we averaged the normalized data and present the results in Figure 3d. The increase of hits was significant at both frequencies, and the increase was larger at 5000 Hz. To test the significance of the effect that sound had on crystallization screening hits, we applied a one-sample t-test to the data. We found that the average ratio of hits with 1000 Hz sound was 1.54, while the average ratio of hits was 1.77 at 5000 Hz. We applied a one-sample t-test to all of the screening data, it showed that the increase of crystallization screening hits using audible sound was significant (P = 0.0003 < 0.05). We also applied audible sound to the crystallization screening of a new target protein. Our preliminary experiments showed promising results. In the crystallization of myocyte enhancer factor 2 (MEF2C), which is a protein associated with muscular atrophy, we found that the protein crystallizes in 1.1 M ammonium titrate dibasic pH 7.0 only under audible sound irradiation at 5000 Hz. We compared the number of crystals obtained with and without audible sound in the reproducibility study by counting the total number of crystals that appeared in 30 droplets under frequencies ranging from 500 to 17500 Hz and in the control without sound. Crystals appeared in all of the droplets and at all frequencies tested, confirming that the crystallization success rate reached 100% at this concentration level, as illustrated in Figure 2d. However, the crystal number varied with the frequencies. Supporting Figure 1 shows that the total number of crystals in the absence of sound was stable at approximately 33, while in the case with audible sound the crystal number varied with the frequency. The number of crystals reached a maximum under 5000 Hz irradiation, confirming a strong effect of audible sound on lysozyme crystallization at this frequency. This experiment demonstrates that crystal nucleation can be affected by audible sound. We also compared the crystal morphologies obtained with or without sound (Supporting Figure 2). Generally, we observed that the crystals obtained under audible sound irradiation show relatively worse morphology than those in the control. The crystals obtained under audible sound were often smaller and have more visible defects on the crystal surface. Crystals in the control more frequently appeared with well-defined faceted surfaces. The crystallization screening and comparative experiments summarized above were carried out at the Northwestern Polytechnical University (NPU) in China. All of the
conditions. At concentration levels between these two extremes, the effect of sound irradiation on the crystallization success rate became obvious (P = 0.0118 < 0.05). Except for the two extreme cases mentioned above, all of the remaining six comparisons showed the same tendency to increase the crystallization success rate when exposed to audible sound. It is likely that crystallization might never occur in solutions with a very low protein concentration because supersaturation is too low. Conversely, at the high concentration level, supersaturation may already be high enough for successful crystallization. Thus, under such extreme conditions, the crystallization success rate becomes insensitive to the effects of sound irradiation. The results from the reproducibility study indicate that audible sound may promote protein crystallization during screening of crystallization conditions. To verify this observation, we carried out a series of crystallization screening studies with and without audible sound irradiation. We examined the crystallization screening of two model proteins (lysozyme and concanavalin A) under eight different sound frequencies ranging from 500−17500 Hz. In Figure 3a, the number of successful crystallization events during screening (hereafter referred as crystallization hits) with audible sound was normalized to the number of hits without sound. The figure shows a similar frequency-dependent trend to that shown in Figure 2b. For both proteins, we observed a maximum in this ratio at a sound frequency of approximately 5000 Hz. To examine the influence of audible sound on the crystallization behavior for other proteins, we arbitrarily selected 15 commercially available proteins and carried out crystallization screening experiments under two specific frequencies (1000 and 5000 Hz). Detailed information on these proteins is shown in Supporting Table 1. The experimental results are shown in Figure 3b and 3c. In general, the crystallization success rate of most of the tested proteins improved when the crystallization trials were exposed to audible sound at both frequencies. In some cases (ribonuclease A I and subtilisin A VII), crystals were only obtained under audible sound. There were a few exceptions (cellulose and pepsin), for which no obvious positive effects were found with irradiation of audible sound. The type of protein is also a variable the influences the effect of audible sound on crystallization. In most cases, sound at a frequency of 5000 Hz produced better screening results than sound at 1000 Hz. To compare the overall effect of sound on the number of crystallization E
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Figure 5. Formation of bubbles under audible sound irradiation. Bubbles appeared after 24 h under 5000 Hz audible sound (a−c) and disappeared after stopping irradiation in another 24 h (a′−c′). Control without sound irradiation after 24 h (d) and 48 h (d′). Hanging-drop method was used for crystallization. Concentrations of lysozyme and NaCl were 20 and 50 mg/mL, respectively.
increases were 1.12, 1.66, 1.74, and 3.67 K under irradiation of 1000, 5000, and 15 000 Hz audible sound and periodic ultrasounds, respectively. According to the results that protein crystallization is affected by sound irradiation, it is likely that the sound may affect the cluster size in the crystallization solution. We used in situ dynamic light scattering (DLS) (XtalController900, XtalConcepts, Hamburg, Germany) to constantly monitor the particle size distribution in the crystallization solution over time with and without sound irradiation (5000 Hz).39 The size distribution is shown in Figure 4c, and the size evolution against time is shown in video 1 (in Supporting Information). Video 1 shows detailed evolution of the hydrodynamic radius of lysozyme particles in solution after the addition of precipitant (NaCl), revealing the influence of audible sound on the crystallization behavior when A) exposed to sound at 5000 Hz; B) without sound irradiation. The hydrodynamic radii are determined by dynamic light scattering and are plotted over time (Video 1, upper left part). The weights of the crystallization droplets were monitored by an ultrasensitive balance and were used to calculate the protein and precipitant concentration during the experiment (Video 1, lower part). Bright-field images of the two droplets were recorded to monitor the macroscopic outcome of the crystallization experiments (Video 1, upper right sections). The acquisition parameters were as follows: interval between images, 300 s; duration of image series, 48 h. Video 1 shows that the cluster size in the crystallization solution with sound irradiation was more homogeneous (size at approximately 1 μm) than that without sound irradiation. This again clearly demonstrates that sound irradiation affects the crystallization process. When crystallization droplets are exposed to the audible sounds, small bubbles may appear as shown in Figures 5a−c. These bubbles did not disappear during the irradiation of audible sound, but did disappear when the sound was stopped (Figure 5a′−c′), indicating that the bubbles were induced by sound. The control experiments showed no detectable bubbles in the droplets (Figure 5d and Figure 5 d′). In addition, most crystals were located in direct proximity to the bubbles (Figure 5a−c). If new crystals appeared after irradiation of audible sound was stopped, they were often found at the locations where bubbles once existed (see the three new crystals shown in Figures 5a′, b′, and c′).
experimental results demonstrate that the audible sound promotes protein crystallization. To further test this effect, similar crystallization screening experiments were carried out in another lab (University of Hamburg (UH) in Germany). An identical experimental setup, resembling the one in NPU was used at UH. Eight proteins were used to analyze the effect of sound at 5000 Hz on crystallization experiments: proteinase K, thaumatin, lysozyme, lipase B, ribonuclease A, carbonic anhydrase, α-glucosidase, and superoxide dismutase. In total, 21 comparative experiments were conducted and all of the results are summarized in Supporting Table 2. It can be seen that in the 21 comparative experiments, 16 showed an increased number of conditions resulting in protein crystals for experiments irradiated with audible sound. We applied a one-sample t-test to the data of the 21 comparative experiments. The evaluation of the experiments confirmed a significantly positive effect of applying audible sound (P = 0.0264 < 0.05). 3.2. Other Phenomena Accompanying Crystallization under Sound Irradiation. During crystallization, sound can induce phenomena that may further influence the crystallization. In this portion of the study, we show that solvent evaporation, temperature, cluster size, and bubble formation in the solution with application of audible sound. To determine whether the sound can enhance the evaporation of the droplets, we tested the amount of evaporation under different sound environments. We used audible sounds at 1000, 5000, and 15000 Hz, as well as a periodic ultrasound where one period = frequency continuously changing from 20 000 to 55 000 Hz over 80 s followed by a quiet interval for 80 s. All of these experiments were conducted at 293 K. Figure 4a shows the results, which indicate that evaporation significantly increased under ultrasound and audible sound. Higher frequency produced a greater amount of evaporation. Visual inspection of the droplet size with and without sound irradiation also indicated the enhancement of evaporation under sound irradiation. We also measured the temperature of the solution when the sound was applied. A thermocouple was used to measure the temperature of 100 μL of deionized water under audible sound and ultrasound treatment at the same location where crystallization occurs. The temperature in the drop was found to increase during the process (Figure 4b), verifying that part of the sound energy was transformed into heat. The temperature F
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break crystal into pieces via harmonic vibration.47 Very loud voices can also shatter materials due to the transmitted vibration energy. In materials processing, vibration caused by sound can break dendrites into pieces. Protein crystals grown in the solution may also be similarly affected. High energy sound may shatter growing protein crystals into small pieces.48 If such sound is applied persistently, no crystals will grow to an observable size.16 Despite these unfavorable factors, our results still show that when sound exposure was persistently applied, favorable crystallization results were obtained. This fact implies that the final crystallization results depend on the combined effects of both favorable and unfavorable factors, and the favorable factors apparently exhibited a stronger influence. To such a combined effect, we selected two factors (the increases in evaporation and temperature) that can be quantitatively estimated and combined them in terms of supersaturation and nucleation rate against sound frequency. The temperature of a solution under irradiation of 1000, 5000, or 15000 Hz audible sound and periodic ultrasound reached 294.12, 294.66, 294.74 and 296.67 K, respectively, in the experimental environment. For these temperatures (293, 294.12, 294.66, 294.74 and 296.67 K), the relationships between the lysozyme solubility (C s ) and the NaCl concentration (CNaCl) were obtained as eqs 1−5, respectively, by curve-fitting the published data.49
4. DISCUSSION The experimental results indicate that protein crystallization is very sensitive to audible sound. In this section, we will discuss the favorable and unfavorable factors for crystallization and then the combined effects arising from audible sound. Finally, we will discuss the complexity of audible sound effects arising from the combination of frequencies and amplitudes. One favorable factor is the enhanced evaporation that can concentrate the crystallization solution, resulting in increased supersaturation when there is no temperature change. Typically, an increase in supersaturation will lead to a higher probability of crystallization. DLS observation (Figure 4c, and Video 1) also revealed a likely favorable factor. At the beginning of the crystallization experiment with lysozyme, the protein solution was in the homogeneous region of the phase diagram. The time constant for the translational diffusion was approximately 100 μs, corresponding to the Brownian motion of lysozyme monomers with a hydrodynamic radius of 1.5 ± 0.3 nm. After addition of precipitant, the hydrodynamic radius increased to approximately 1 μm. This fraction might represent metastable mesoscopic clusters of lysozyme molecules.40,41 The formation of these dense liquid clusters occurs very rapidly after addition of the precipitant and their accumulation in the crystallization experiment can be followed by in situ DLS (see Video 1 for a comparison of in situ observation of the size evolution over time, with and without sound irradiation). The clusters are in constant equilibrium with monomeric lysozyme in the bulk solution. If the dense liquid is stable over time, nucleation of crystals occurs inside the clusters.42,43 When applying sound irradiation, the size range of these clusters in the crystallization solution was clearly narrower than that observed without sound irradiation (Figure 4c). The formation of a periodic crystal within a dense liquid protein cluster is the rate limiting process in nucleation;40 therefore, the observed difference in the size of these clusters might be an explanation for the substantial influence of audible sound on crystallization experiments. Another favorable factor is the formation of bubbles that arise when the sound is applied (Figure 5). The formation of bubbles is similar to the “cavitation” effect of ultrasound irradiation44,45 and may be an important factor for increasing the nucleation of crystals. In this study, the formation of bubbles may have promoted crystallization by two possible mechanisms: (1) the bubble surface may provide a nucleation surface that lowers the energy barrier for nucleation or, (2) when the bubble breaks, the breakage may provide a localized high concentration of protein and salt, which overcomes the nucleation barrier to form a nucleus. Once a nucleus is formed, crystal growth will become a spontaneous process as increasing the size of the crystal results in a reduced free energy (i.e., ΔG < 0). This mechanism may be one possible reason for the increased chance of obtaining crystals. Apart from the above-mentioned favorable factors, there are also some unfavorable factors that may affect protein crystallization. One of these is the increase in temperature due to the sound irradiation (Figure 4b). Most proteins have a higher solubility at higher temperature, meaning an increase in temperature is expected to result in a decrease in supersaturation and an associated reduction in the chance of obtaining crystals.46 Another unfavorable factor may be that the crystals are shattered into small pieces by sound, and audible sound can
Cs = 0.00011C NaCl 3 − 0.00073C NaCl 2 − 0.16256C NaCl (1)
+ 3.9819
Cs = −0.00094C NaCl 3 + 0.1488C NaCl 2 − 7.6268C NaCl (2)
+ 129.9071 Cs = −0.00094C NaCl 3 + 0.1493C NaCl 2 − 7.7C NaCl
(3)
+ 132.3466 Cs = −0.00094C NaCl 3 + 0.1494C NaCl 2 − 7.7114C NaCl
(4)
+ 132.7216 Cs = −0.00094C NaCl 3 + 0.1524C NaCl 2 − 8.0315C NaCl + 142.8652
(5)
The above equations and the evaporated amounts under different sound environments can be used to roughly estimate the supersaturation (defined as σ = C/Cs, where C is the protein concentration and Cs is the solubility) in the solution under different conditions when the initial lysozyme and NaCl concentrations were 15 and 30 mg/mL, respectively. The nucleation rate I can be calculated from the lysozyme concentration and its solubility as ⎛ C − Cs ⎞a I = k n⎜ ⎟ ⎝ Cs ⎠
(6)
where kn and a are constants. As proposed by Saikumar et al., the nucleation rate can be calculated as follows:50 ⎛ C − Cs ⎞3 I = 7.71 × 10−2 × ⎜ ⎟ ⎝ Cs ⎠
(7)
The results (Figure 6a) show that sound irradiation increases the supersaturation in a frequency-dependent manner. SuperG
DOI: 10.1021/acs.cgd.5b01268 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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AUTHOR INFORMATION
Corresponding Authors
*Phone: 040-42838-3123/6069. Fax: 040-42838-7818. E-mail:
[email protected]. *Phone: +86-29-88460254. Fax: +81-29-88460254. E-mail:
[email protected]. Author Contributions
C.Y.Z. and Y.W. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. D.C.Y. designed research; C.Y.Z., Y.W., R.S., Y.L., M.Y.W., Y.M.L., Y.Z.G., and Z.Q.W. performed research; C.Y.Z., R.S., D.C., C.D., H.M.L., Y.M.L., C.B., and D.C.Y. analyzed data; and C.Y.Z., R.S., C.B., and D.C.Y. wrote the paper.
Figure 6. Estimated supersaturation and nucleation rates under different sound frequencies. (a) Supersaturation and (b) nucleation rates were estimated by monitoring the solvent evaporation and temperature increases during crystallization.
Notes
The authors declare no competing financial interest.
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saturation reaches its maximum value at a frequency of 15 000 Hz, while this value decreases sharply under ultrasound. Because supersaturation is the driving force of crystal nucleation and growth, the nucleation rates in different treatment groups were calculated. Figure 6b shows the nucleation rate results. The nucleation rates under different sound treatment showed the same tendency as that of supersaturation. Because the nucleation rate is positively correlated with the crystallization success rate, these estimated results also confirm that the crystallization success rate is frequency-dependent. In the experiment, the maximum hit was observed at 5000 Hz in Figures 2 and 3, which is different from the results shown in Figure 6. However, we believe this is reasonable because in the estimation, we only considered two factors, while the real factors contributing to the overall effect are likely to be much more complicated.
ACKNOWLEDGMENTS This work was supported by National Basic Research Program of China (973 Program, Grant No. 2011CB710905), National Natural Science Foundation of China (Grant No. 31170816, 11202167, 31402019), China Postdoctoral Science Foundation (Grant No. 2013T60890), and FRF (the Fundamental Research Foundation, 3102014JKY15006) of NPU in China. Undergraduate Training Programs for Innovation and Entrepreneurship in China (Grant No. 201410699087). The investigators R.S. and C.B. were supported by the excellence cluster “The Hamburg Centre for Ultrafast Imaging Structure, Dynamics and Control of Matter at the Atomic Scale” of the Deutsche Forschungs-gemeinschaft (DFG) and by the Röntgen-Angström-Cluster (project 05K12GU3) funded by the German Federal Ministry of Education and Research (BMBF). The authors in NPU would also like to thank NPU Graduate Student Innovation Center for the help in providing part of the experimental conditions.
5. CONCLUSIONS Audible sound commonly exists throughout many processes, such as crystallization, melting, solidification, dissolution, condensation, and other changes in material states. In most of these processes, audible sound is typically not considered as a parameter that may change the final result. In protein crystallization, researchers often ignore the potential effects of audible sound on protein crystallization. Here, we investigated the effect of audible sound parameters on protein crystallization, especially frequency. We demonstrate that the protein crystallization success rate increased significantly when sound frequency was set at 5000 Hz, and the mechanism was also tried to clarify. This discovery can help to improve protein crystallization by intentionally supplying an audible sound in the environment.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b01268. Crystal number and examples of crystal morphology with and without audible sound, proteins and their buffers used in the investigation, and number of crystallization hits with and without sound irradiation in verification study in University of Hamburg (PDF) DLS monitoring of the particle size evolution against time with and without sound irradiation (AVI) H
DOI: 10.1021/acs.cgd.5b01268 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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