Protein crystallization irradiated by audible sound: the effect of varying

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Protein crystallization irradiated by audible sound: the effect of varying sound frequency Chen-Yan Zhang, Jie Liu, Meng-Ying Wang, Wen-Jing Liu, Nan Jia, ChangQing Yang, Ming-Liang Hu, Yi Liu, Xian-Yu Ye, Ren-Bin Zhou, and Da-Chuan Yin Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b01339 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 10, 2018

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Crystal Growth & Design

Protein crystallization irradiated by audible sound: the effect of varying sound frequency Chen-Yan Zhang†, *, Jie Liu†, Meng-Ying Wang†, Wen-Jing Liu, Nan Jia, Chang-Qing Yang, Ming-Liang Hu, Yi Liu, Xian-Yu Ye, Ren-Bin Zhou, 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

Shenzhen Research Institute of Northwestern Polytechnical University, Shenzhen 518057, PR China

The authors declare no conflict of interest. * To whom correspondence may be addressed. †

The contribution of these authors was equal.

Chen-Yan Zhang, Email: [email protected], Tel: +86-29-88460543; Da-Chuan Yin, Email: [email protected], Tel: +86-29-88460254.

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Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract Protein crystallization is a process that is very sensitive to the physical environment. Audible sound is an environmental characteristic that can significantly affect the crystallization process. Previously, it was found that the crystallization result is frequency dependent, i.e., the crystallization of protein under different sound frequencies yields different results. Here, we further investigate the effect of varying frequency (or a frequency program) on protein crystallization. Twelve different frequency programs and six proteins were used to test the effect of varying sound frequency on protein crystallization. The results showed that varying the audible sound frequency from high to low exhibited the most significant improvement in protein crystallization. Varying frequency linearly from 15,000 Hz to 100 Hz in 12 h best promoted crystallization, with the average number of crystallization hits 36.5% higher than in the control. Crystal quality was improved with sound irradiation using STW 2 program. Our study showed that varying the sound frequency have positive effects on facilitating protein crystallization, and purposely optimizing the sound frequency programs may be helpful for obtaining protein crystals.

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1. Introduction Protein crystallization is an important process for applications such as the mass production of proteins for pharmaceutical1, food and biochemical industries2, determination of three-dimensional structures of protein molecules, and creation of ideal model systems for mechanistic study of crystal nucleation and growth3,4. The practice of protein crystallization, however, is often considered troublesome due to the difficulties in finding chemicals or combinations of chemicals that can help crystallize the target proteins (hence the need for so-called crystallization screening)5-9 and in optimizing the crystallization conditions to obtain high-quality protein crystals (crystallization optimization)10,11. Even though huge amounts of effort have been devoted to this field12-14, more studies to facilitate crystallization screening and optimization are still urgently needed. Since protein crystallization is highly sensitive to environmental factors (sound, light, electric and magnetic fields, gravitational field, heat, mechanical vibration, and so on)15-20, investigation of the effects of the environments on protein crystallization is essential for good control of the crystallization process to produce desirable results. Audible sound has been shown to be an environmental factor that affects the process of protein crystallization21. We used real-world sound and evaluated its effect on protein crystallization. The correlation between sound parameters and the crystallization success rate was studied mathematically, and it was found that crystallization was dependent on the frequency, amplitude, volume, irradiation time, overall energy and spectral characteristics of the sound15. Sound frequency was a particularly important parameter15,21. Protein crystallization was dependent on the frequency of the audible sound. A fixed frequency of 5,000 Hz yielded the most significant enhancement in crystallization success rate and crystallization screening hits21. Based on the previous results, we anticipated that protein crystallization could also be affected by a sound environment in which the frequency constantly varies. 3



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Hence, we conducted further investigation on the effect of a varying frequency program on protein crystallization. We selected twelve different frequency programs and tested crystallization of six different proteins under these programs. The data confirmed that varying sound frequency could indeed affect protein crystallization, and different frequency programs showed different power in facilitating the crystallization process. The possible mechanisms are also discussed. Our results indicated that, it is possible to enhance protein crystallization by deliberately applying a sound program.

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2. Materials and methods 2.1. Materials The proteins used in this study were all commercially available and utilized without further purification (detailed protein information is listed in Table 1). IndexTM crystallization screening kits were obtained from Hampton Research Co. (Aliso Viejo, CA, USA). The chemical reagents sodium chloride, sodium acetate trihydrate and HEPES-Na were purchased from Chemical Reagent Co. (Shanghai, China), Xi'an Chemical Reagent Factory (Xi'an, China), and Shanghai Kayon Biological Technology Co. (Shanghai, China), respectively. Table 1. Detailed information of proteins used in this study proteins

abbreviations

lysozyme concanavalin A α-chymotrypsinogen catalase proteinase K

lys. con. chy. cat. prok.

glucose isomerase

glu.

companies

buffers

final concentrations (mg/ml)

Seikagaku Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Hampton Research

0.1 M NaAc, pH 4.6 0.1 M HEPES-Na, pH 7.0 0.1 M HEPES-Na, pH 7.0 0.1 M HEPES-Na, pH 7.0 0.1 M HEPES-Na, pH 7.0

10 10 10 7.5 7.5

0.1 M HEPES-Na, pH 7.0

3.5

2.2. Experimental setup The experimental setups we used in this study were the same as described previously21. One of the setups included a temperature-controlled chamber with a loudspeaker installed inside the chamber, and the other did not have a loudspeaker installed. These setups were used as the experimental (with the loudspeaker) and control groups (without the loudspeaker). For the experiment setups, the loudspeaker was installed just right above the crystallization plate, and sound wave was propagated to one direction, and in this case loud speaker could cover the whole crystallization plate. The intensity of sound was measured by using sound pressure meter (PHONIC PAA3X, USA), and it was changed with sound frequency. The 5



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intensity of sound was 82 dB when frequency was 1000 Hz, the intensity of sound was 87 dB when frequency was 5000 Hz, the intensity of sound was 82 dB when frequency was 10000 Hz, and the intensity of sound was 64 dB when frequency was 15000 Hz. The temperature in both groups was calibrated to be the same with  0.1 K accuracy, when the sound was being played to make sure that the crystallization conditions were the same except for the sound environment. The loudspeaker was connected to a sound-control system, which could vary the sound frequency between 20 Hz to 20000 Hz. The temperature-controlled chambers were wrapped with sound insulation cotton (Owens Corning, Shanghai, China) and deadening felt (Sanchez Goma G, Guangzhou, China) to prevent external sound penetration. A record pen (Meizu, Zhuhai, China) was used to record the sound in the experiment incubators, which was analyzed by Cool Edit Pro 2.0 software (Adobe Systems, California, USA) to ensure accurate control of the sound frequency in the experiment group21. 2.3. Crystallization Experiments Three series of experiments were designed to test the effects of twelve sound frequency programs on protein crystallization including (1) the protein crystallization reproducibility, (2) the protein crystallization screening, and (3) the mechanism study. 2.3.1. The crystallization reproducibility study We used the crystallization success rate as the indicator to demonstrate the effect of the sound frequency program on the protein crystallization. We used lysozyme as a model protein to test the crystallization reproducibility with irradiation by different programs. (1) Figure 1a1-1a4 shows four types of triangle wave (TW) frequency programs. In program TW 1 (Fig. 1a1), the sound frequency increased linearly from 100 Hz to 15000 Hz and then decreased to 100 Hz in a period of 24 h. In program TW 2 (Fig. 1a2), the sound frequency decreased linearly from 15000 Hz to 100 Hz and then 6


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increased to 15000 Hz in a period of 24 h. Program TW 3 (Fig. 1a3) was the same as program TW 1 except that the period was 48 h. Program TW 4 (Fig. 1a4) was the same as program TW 2, except that the period was 48 h. (2) Figure 1b1-1b4 shows four types of sawtooth wave (STW) frequency programs. In programs STW 1 (Fig. 1b1) and STW 3 (Fig. 1b3), the sound frequency increased from 100 Hz to 15000 Hz linearly in a period of 12 h and 24 h, respectively. In programs STW 2 (Fig. 1b2) and STW 4 (Fig. 1b4), the sound frequency decreased from 15000 Hz to 100 Hz linearly in a period of 12 h and 24 h, respectively. (3) Figure 1c1-1c4 shows four types of square wave (SW) frequency programs. In program SW 1 (Fig. 1c1), with increasing frequency, the frequency was held at 1000 Hz, 5000 Hz, 10000 Hz and 15000 Hz for 12 h each. In program SW 2 (Fig. 1c2), with decreasing frequency, the frequency held at 15000 Hz, 10000 Hz, 5000 Hz and 1000 Hz for 12 h each. In program SW 3 (Fig. 1c3), the frequency was set consecutively at 1000 Hz, 15000 Hz and 1000 Hz for 12 h, 24 h and 12 h, respectively. In program SW 4 (Fig. 1c4), the frequency was set consecutively at 15000 Hz, 1000 Hz and 15000 Hz for 12 h, 24 h and 12 h, respectively.

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Figure 1. Three different sound frequency programs. Triangle wave (TW) schematic diagrams: (a): 100 Hz -15000 Hz -100 Hz over a period of 24 h (a1) for program 1 and 48 h (a3) for program 3; 15000 Hz -100 Hz -15000 Hz over a period of 24 h (a2) for program 2 and 48 h (a4) for program 4. Sawtooth wave (STW) schematic diagrams: (b): 15000 Hz-100 Hz over a period of 12 h (b2) for program 2 and 24 h (b4) for program 4, 100 Hz-15000 Hz over a period of 12 h (b1) for program 1 and 24 h (b3) for program 3. Square wave (SW) schematic diagrams (c): (c1) the frequency remained at 1000 Hz, 5000 Hz, 10000 Hz, and 15000 Hz for 12 h each; (c2) the frequency remained at 15000 Hz, 10000 Hz, 5000 Hz, and 1000 Hz for 12 h each; (c3) the frequency remained at 1000 Hz, 15000 Hz, and 1000 Hz for 12 h, 24 h and 12 h; (c4) the frequency remained at 15000 Hz, 1000 Hz, and 15000 Hz for 12 h, 24 h and 12 h.

Lysozyme (15 mg/ml and 20 mg/ml) and NaCl (30 mg/ml) were used for the test. The sitting drop method was utilized, and each crystallization droplet was set up by mixing 1 μl protein solution with 1 μl crystallization agent; the volume of the reservoir solution was 80 μl; the crystallization solution was dispensed into 96-well 8


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Intelli-Plate crystallization plates (Hampton Research, California, USA), and a crystallization robot (Screenmaker 96 + 8TM, Innovadyne Technologies Inc., California, USA) was used to set up the crystallization trials. Crystal Clear Sealing Tape (Hampton Research, California, USA) was used to seal the crystallization plates. The crystallization plates were then placed into temperature-controlled chambers with (experimental group) and without (control group) sound irradiation. After incubation for 48 h, an automated crystal image reader (XtalFinder XtalQuest Inc., Beijing, China) was used to capture and record the crystallization images. Thus, the crystallization success rate (defined as the ratio of the number of crystallization droplets that yield crystals to the total number of crystallization droplets) could be calculated. 2.3.2. Crystallization screening study Based on the results of the crystallization reproducibility study, we selected the most efficient sound frequency program, STW 2, for the crystallization screening study. Two sets of protein crystallization screening experiments were carried out: 1) comparison of crystallization screening hits obtained with the most efficient frequency program (STW 2 group) and the control (no sound group); 2) comparison of crystallization screening hits obtained with the most efficient frequency program (STW 2 group) and the audible sound at 5000 Hz. Audible sound at a monotonous frequency at 5000 Hz was used, because it was found to be the most effective monotonous sound frequency for facilitating protein crystallization, in the previous study21. We used six commercially available proteins as the model proteins for the screening study: lysozyme, concanavalin A, α-chymotrypsinogen, catalase, proteinase K, and glucose isomerase (detailed information of these proteins is listed in Table 1). IndexTM crystallization screening kits were used as the crystallization agents, and the process to set up the crystallization trials was the same as that used for the crystallization reproducibility study. The crystallization reagents used in Figure 5 are 9



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as followed: all the crystallization reagents were from IndexTM screening kits of Hampton Research Co., the crystallization reagent of glucose isomerase is No. G12 (0.2 M magnesium chloride hexahydrate, 0.1 M HEPES pH 7.5, 25% w/v polyethylene glycol 3,350); the crystallization reagent of catalase is No. H3 (0.2 M sodium malonate pH 7.0, 20% w/v polyethylene glycol 3,350); the crystallization reagent of proteinase K is No. A7 (0.1 M citric acid pH 3.5, 3.0 M sodium chloride); the crystallization reagent of a-chymotrypsinogen is No. A7 (0.1 M citric acid pH 3.5, 3.0 M sodium chloride); the crystallization reagent of concanavalin A is No. H4 (0.2 M ammonium citrate tribasic pH 7.0, 20% w/v polyethylene glycol 3,350); the crystallization reagent of lysozyme is No. H3 (0.2 M sodium malonate pH 7.0, 20% w/v polyethylene glycol 3,350). 2.3.3. The variation of protein concentration under the irradiation of audible sound with different frequency programs We previously found that irradiation with audible sound can increase the temperature of the sample21, resulting in variation in supersaturation, which affects crystallization. In this research, the irradiation of sound could certainly affect the temperature, result in affecting crystallization result. To explore the mechanism of the effect of audible sound with different frequency programs, the temperature variation and concentration variation under the influence of the sound were examined. We measured the temperature of the crystallization droplet and calculated the volume variation of crystallization droplet to obtain the supersaturation and nucleation rate under sound irradiation at different frequency programs. Three frequency programs, STW 2 (15000 - 100 Hz, 12 h period), STW 1 (100 15000 Hz, 12 h period), 5000 Hz audible sound, as well as no sound group. The measurement setup is shown in Figure 2, and the detailed procedure was described previously22. A hanging drop model was used for the experiment. The hanging crystallization droplet was dispensed on a cover glass (the lower cover glass, ϕ 6 mm) connected with another larger one (the upper glass, 20 mm × 20 mm) using a spacer 10


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(the Plexiglass gasket, 5 mm × 5 mm). The hanging crystallization droplet was sealed with the reservoir solution (80 μl, 60 mg/ml NaCl) in an ELISA strip (FEP100012, Guangzhou Jet BioFiltration Product Co., Guangzhou, China) using high vacuum grease and a 20 mm × 20 mm rectangular cover glass. The spacer was used to avoid blocking the optical path with the upper edge of the well and the vacuum grease. The crystallization droplet was prepared by mixing 1 μl 60 mg/ml NaCl with 1 μl 40 mg/ml lysozyme. The whole setup was placed into the 293 K temperature-controlled chamber connected with a programmable water circulator (Polyscience 9712 refrigerated circulator, Polyscience Inc., Pennsylvania, USA). The images of the crystallization droplets were recorded every two hours by polarization microscope (Olympus SZX2-ILLB, Shanghai, China) equipped with a digital camera (Cannon EOS 550D, Beijing, China), and the temperature of the droplets was measured by a temperature sensor (Sartorius BS124S, Gottingen, Germany) every two hours until twelve hours after setting up the crystallization droplets. Then, the values of r and h (Figure 2) of the crystallization droplets were obtained from the images using software Adobe Photoshop CS6 (Adobe, California, USA). The volume of crystallization droplet (Vt) was calculated with Equation (1). Vt = πh (3r2 + h2) / 6

(1)

Figure 2. The setup for the measurement of droplet volume. In (a), 1: upper cover glass; 2: Plexiglass gasket; 3: lower cover glass; 4: crystallization droplet; 5: reservoir solution. In (b), 1: cover glass; 2: crystallization droplet.

2.4. Crystal quality and structure comparisons with and without sound irradiation 11



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We compared the crystal quality and structure with and without sound irradiation using lysozyme and proteinase K as model proteins. The crystallization conditions (after mixing) for the two proteins were: for crystallization of lysozyme: 7.5 mg/ml lysozyme, 80 mg/ml NaCl; for crystallization of proteinase K: 10 mg/ml proteinase K, 158.57 mg/ml (NH4)2SO4. Sitting-drop method was used. The crystals were grown at 293 K for 48 h, with and without sound irradiation using the program STW 2. Crystals with similar size were harvested with nylon loops (Hampton Research Co., USA). For each group, at least five crystals were used for the comparisons. All the crystals were soaked rapidly in a cryoprotectant solution, i.e., the mixed solution of the corresponding crystallization reagent (75%) and glycerol (25%). The diffraction data were collected at 100 K in nitrogen stream by using Macromolecular Crystallography Beamline (BL17U1) with ADSC Quantum 315r CCD detector at the Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China). Data processing and scaling were performed by HKL 3000 software package23. The 3D structure of lysozyme were resolved by molecular replacement with CCP4 software for the comparison24, and the structure of lysozyme (PDB ID 2CDS) was used as the model. The program Coot was used to rebuild the initial model25. The superimposed comparison was analyzed using PyMOL software package26 and TM-align ((https://zhanglab.ccmb.med.umich.edu/TM-align/).

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3. Results 3.1. Crystallization reproducibility study Figure 3 shows the results of the crystallization reproducibility study for lysozyme. As shown in the figure, we used the normalized crystallization success rate to the control as the standard for easy comparison. It can be seen that, in all cases, the crystallization success rate was higher when the audible sound was applied, and regardless of which sound program was used. In the case of triangle waves (see Figure 3a), it can be seen that, in addition to the enhancement in the crystallization success rate by applying the sounds, there is another phenomenon worthy of further examination. Wave programs TW 2 and TW 4 showed a more prominent effect than the other two programs (TW 1 and TW 3). We noticed that the common feature of TW 2 and TW 4 was that both programs varied the frequency from high to low (from 15000 Hz to 100 Hz), while the other two programs (TW 1 and TW 3) varied the frequency from low to high (100 Hz to 15000 Hz). Use of the TW 4 program resulted in the highest increase in crystallization success rate compared with the control at 1.4 times higher. In the case of sawtooth waves (see Figure 3b), we also observed enhancement of the crystallization success rate by applying audible sound irradiation. Among the four programs, STW 2 showed the most obvious effect. At initial concentrations 15 mg/ml and 20 mg/ml, the crystallization success rate under sound irradiation was 4.45 and 3.91 times than that without sound irradiation, respectively. Another notable phenomenon was that, at lower initial protein concentration (lysozyme: 15 mg/ml), the trend was more obvious. In the case of square waves (see Figure 3c), we again observed improvement in the crystallization success rate. Among the four different programs, SW 2 showed the most obvious effect. In this experiment, we also noted that the improvement in the crystallization success rate was more obvious at lower initial protein concentration (15 mg/ml). 13



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After careful examination, we noticed another interesting phenomenon: for all of the experiments, the crystallization success rate tended to be higher when the sound frequency moved from high to low (from 15000 Hz to 100 Hz, as in programs TW 2, TW 4, STW 2, STW 4, SW 2 and SW 4). To further test whether this tendency was significant, a statistical analysis was carried out. The twelve frequency programs were classified into two groups: (1) group 1: those starting from the low frequency (100 Hz) and (2) group 2: those starting from the high frequency (15000 Hz). Figure 3d shows that both groups showed enhancement of the crystallization success rate, and the hit improvement increased from 167.17% in group 1 to 212% in group 2 (p = 0.0087 < 0.01), so group 2 showed a higher success rate compared with group 1. For 15 mg/ml lysozyme, the crystallization success rate increased more significantly in group 2 (p = 0.0431 < 0.05). In addition, at the lower initial concentration (15 mg/ml), the improvement in the crystallization success rate (217.58%) was higher than at the higher initial concentration (20 mg/ml) (161.58%, p = 0.0195 < 0.05).

Figure 3. Crystallization success rate with and without sound irradiation for the different frequency programs. (a) Triangle wave programs (programs TW 1 - TW 4); (b) sawtooth wave

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programs (programs STW1 - STW4); (c) square wave programs (programs SW 1 - SW 4); (d) two groups of sound programs: 1. the group starting from the low frequency (100 Hz); 2. the group starting from the high frequency (15000 Hz). The initial crystallization solution conditions were 15 mg/ml or 20 mg/ml lysozyme and 30 mg/ml NaCl. The difference between program 1 and program 2 was significant for 15 mg/ml lysozyme (p = 0.0431 ˂ 0.05), and the improvement of hits between the control and 15 mg/ml lysozyme in frequency program 2 was significant (p = 0.0436 ˂ 0.05). The sound intensity level was set to -5 dB for all groups.

3.2. Crystallization screening study As shown above, program STW 2 showed the best performance in the crystallization reproducibility study of lysozyme. We selected this program for the crystallization screening study. Figure 4a shows the results. Six proteins (lysozyme, concanavalin A, catalase, glucose isomerase, proteinase K, and α-chymotrypsinogen) were selected as the model proteins, with detailed information listed in Table 1. From Figure 4a, it can be seen that program STW 2, as expected, increased the crystallization screening hits compared with the control. The increases in the crystallization screening hits were 23%, 34%, 19%, 20%, 48% and 75% for lysozyme, concanavalin A, catalase, glucose isomerase, proteinase K, and α-chymotrypsinogen, respectively. Figure 4b shows the average crystallization screening hits with and without program STW 2 based on the data presented in Figure 4a. The average increase in the crystallization screening hits was 32% when using program STW 2 compared with the control (p = 0.0258 < 0.05). The image of crystallization droplets during crystallization screening are shown in Figure 5. The crystal number increased significantly with sound irradiation via program STW 2 compared with control, as proteinase K in Figure 5c and Figure 5c´, α-chymotrypsinogen in Figure 5d and Figure 5d´, lysozyme in Figure 5f and Figure 5f´.

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Figure 4. Crystallization screening hits with and without audible sound. (a) The results of the crystallization screening hits with and without program STW 2. (b) Normalized average of hits with and without the audible sound program STW 2. 1: no sound group and 2: STW 2. The sound intensity level was set to -5 dB for all groups. Lysozyme was abbreviated as lys., concanavalin A was abbreviated as con., catalase was abbreviated as cat., glucose isomerase was abbreviated as glu., proteinase K was abbreviated as prok., and α-chymotrypsinogen was abbreviated as chy.

Figure 5. Image of crystallization droplets in control (a - f) and STW 2 group (a´- f´). a and a´ are glucose isomerase, b and b´ are catalase, c and c´ are proteinase K, d and d´ are α-chymotrypsinogen, e and e´ are concanavalin A, f and f´ are lysozyme, The crystallization reagents used here for each protein have been listed in Methods part.

In our previous research, we found that monotonous frequency 5000 Hz showed the best ability to increase the crystallization screening hits21. Here, we compared the 16


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screening hits using a monotonous frequency at 5000 Hz and program STW 2. We arbitrarily selected four proteins for the comparison. The results are shown in Figure 6a. Both the monotonous 5000 Hz frequency and program STW 2 increased the crystallization screening hits, and the increase was similar for both sounds. Among the four proteins, glucose isomerase showed the largest increase in screening hits using program STW 2 compared to 5000 Hz. For the rest proteins, the increase by using the two sounds showed comparable results. Monotonous sound at 5000 Hz and program STW 2 can enhance protein crystallization to a similar level, although there is some variation based on the protein type.

Figure 6. Crystallization screening hits of proteins under the influence of two different sounds: monotonous frequency at 5000 Hz and program STW 2. (a) Comparison of screening hits in three groups: no sound, 5000 Hz and the STW 2 program. (b) Statistical comparison of the screening hits in three groups. The sound intensity level was set to -5 dB for 5000 Hz and STW 2 group.

3.3. Variation of the volume and temperature of the crystallization droplet In our previous study, it was reported that the actual evaporation volume and temperature of the droplets increased under irritation with fixed frequency audible sound21. One potential explanation is that the energy produced by audible sound was absorbed by crystallization droplets, so both the temperature and evaporation increased. 17



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The sound wave may affect the evaporation of water (and other volatile chemicals in the solution) during the crystallization process, thus affecting the crystallization. To find out if it is true we measured the volume of the droplet and temperature during the course of applying the sound wave at different frequency programs, including the control, 5000 Hz, STW 2, and STW 1. The measurement setup was shown in Figure 2, and the method was presented elsewhere22. The crystallization droplet was mixed by 1 μl 60 mg/ml NaCl with 1 μl 40 mg/ml lysozyme. Their evaporation and temperature was measured every two hours for a total of twelve hours under irradiation by different sound programs. Figure 7 shows the results. The drop volume decreased under all sound programs; it decreased the most under the STW 2 program, followed by the STW 1 program and then the 5000 Hz program. The volume of the droplets in the control group was almost constant. Additionally, the temperature of the droplets changed with irradiation by different sound programs, consistent with a previous study21. The final temperature was 293.06 K in the control, 294.66 K in the 5000 Hz group, 294.6 K in the STW 2 group, and 294.7 K in the STW 1 group. Therefore, both the volume and temperature of the crystallization droplets were affected by the sound programs, although different sound programs had different effects on the variation of droplet volume and temperature.

Figure 7. The effect of audible sound frequency programs on variation of the droplet volume (a) and temperature using different programs (b). The temperature and volume were measured every two hours after setting up the crystallization droplets for twelve hours with irradiation of different sound programs, including STW 2, STW 1, and 5000 Hz.

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3.4. Comparison of crystal quality and 3D structure with and without STW 2 The sound irradiation using different sound frequency programs showed significant effect in improving protein crystallization in both reproducibility and screening studies. Among the programs, STW 2 showed the best performance in promoting crystallization. So we used this program to examine if the sound irradiation affects crystal quality and molecular structure or not. The results were shown in Figure 8. More detailed data is listed in the Supplementary part in Tables S1 and S2. From the results, it could be seen that, the resolution limit of both lysozyme and proteinase K crystals was improved significantly under sound irradiation. For lysozyme crystals, the average resolution limit was improved from 1.618 Å for no sound group, to 1.352 Å for STW 2 group (p = 0.0207 < 0.05); for proteinase K crystals, the average resolution limit was improved from 1.648 Å for no sound group, to 1.348 Å for STW 2 group (p = 0.0106 < 0.05). The average mosaicity of lysozyme crystals was improved from 0.588° in no sound group to 0.316° in STW 2 group (p = 0.0298 < 0.05)；for proteinase K crystals, it was improved from 0.308° in no sound group to 0.1° in STW 2 group. The B factor was also improved from 18.46 in no sound group to 14.7 in STW 2 group for lysozyme crystals; and from 6.78 in no sound group to 4.2 in STW 2 group for proteinase K crystals. For lysozyme crystals, the average I/σI was 2.13 and 3.49 in control and STW 2 group, respectively; For proteinase K crystals, the average I/σI was 8.626 and 16.318 in control and STW 2 group (p = 0.0161 < 0.05), respectively. These results demonstrated that the crystal quality of proteins could be indeed improved significantly by using sound irradiation, which was in good agreement with our previous results15. To examine if the sound irradiation affects the molecular structure or not, we compared the 3D structures of lysozyme molecules resolved using crystals obtained with and without sound irradiation, using PyMOL software and TM-align. The resolved and refined 3D structures are shown in Figure 9a for lysozyme in no sound group, Figure 9b for lysozyme in STW 2 group, and Figure 9c for superimposed 19



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contrast results. The results showed that similarity of 3D lysozyme structure with and without STW 2 was 99.92% by using TM-align, and the root-mean-square deviation (RMSD) value for the structure comparison was 0.106. These comparisons showed that the 3D structure of lysozyme in both groups was almost identical. Hence we concluded that there was no detectable structure difference caused by sound irradiation.

Figure 8. Comparison of diffraction data of crystals grown with and without sound irradiation using STW 2 program. (a) Resolution limits (p = 0.0207 < 0.05 for lysozyme; p = 0.0106 < 0.05 for proteinase K); (b) Mosaicity (p = 0.0298 < 0.05 for lysozyme); (c) B factor; (d) I/σI (p = 0.0161 < 0.05).

Figure 9. Comparison of 3D structure of lysozyme molecules with and without STW 2. (a)

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Lysozyme 3D structure in no sound group; (b) Lysozyme 3D structure in STW 2 group; (c) Superimposed contrast results.

In our previous work21, we monitored the size evolution by in situ dynamic light scattering in 5000 Hz and no sound group. We have found that, there was an obvious difference in that the cluster size in the initial stage with sound irradiation was much more homogeneous than that without sound irradiation. It means that the dense liquid phases after the phase separation were homogenized by the sound. This was a very good indication that the nucleation process was affected by the sound irradiation. Further, the crystal quality improvement as proved by the X-ray diffraction analysis indicated that the nuclei maybe better than the normal case. In fact we think that the sound irradiation may remove some impurities and wrongly packed protein molecules away from the packing site after they are energized by the sound, so that those correctly packed molecules (correct molecule, correct site, and correct direction) can pack more perfectly. If this mechanism is correct, it will affect the crystal packing in the growth process, and finally affect the crystal quality.

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4. Discussions In this work, we investigated the effect of varying frequency of audible sound on protein crystallization. Similar to the effect of fixed frequency, audible sound with varying frequency also showed a positive effect on promoting protein crystallization. In our past work, we proposed that crystallization was affected by sound due to the complicated effects of sound on the evaporation of solvent, temperature, cluster size and bubble formation, etc.21. Some of these effects (for example, evaporation of the solvent, bubble formation, and formation of more homogeneous clusters) are favorable for crystallization, but others (like increased temperature and shattering of crystals by sound) are unfavorable. The final crystallization results are determined by the combined effects of these factors. We have also shown that the two effects, i.e., the increase in the temperature and the evaporation of the solvent, can be quantified and used to estimate the supersaturation in the solution. From the supersaturation, the nucleation rate can also be estimated, providing a rational explanation of the phenomenon that there is an optimal frequency for facilitating crystallization. In our current study, the effects of applying sound were similar to those found when fixed frequency sound was applied, i.e., effects such as temperature increase, solvent evaporation, etc. also occur when varying frequency sound was used. We confirmed these phenomena experimentally by measuring the drop volume and temperature. The change in measured volume indicated that solvent evaporation was indeed facilitated by applying sound, and the increase in temperature also confirmed that sound can affect the temperature of the solution. Based on the measured results, we estimated supersaturation and nucleation rate under the influence of sound with varying frequency. We measured the variation of the drop volume over time during the course of applying the sound programs (STW 1 and STW 2) or 5000 Hz audible sound, and we simultaneously recorded the temperature throughout the whole process. Figure 7 shows the measurement of the drop volume and temperature in the course of applying 22


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the sound; the final temperature of the drop was 293.06 K in the control, 294.66 K in 5000 Hz, 294.6 K in the STW 2 program, and 294.7 K in the STW 1 program after applying sound for 12 h. By curve-fitting published data27, we obtained the relationships between the solubility (Cs) of lysozyme and the concentration of NaCl (CNaCl) at different temperatures, represented by Equations (2) - (5), corresponding to the different temperatures in different groups: Cs = -0.00086CNaCl3 + 0.13CNaCl2 - 6.90CNaCl+ 118.10

(control)

(2)

Cs = -0.00104CNaCl3 + 0.16CNaCl2 – 8.34CNaCl + 141.44

(STW 2)

(3)


(5000 Hz)

(4)


(STW 1)

(5)

The supersaturation, σ, can be calculated according to the relationship σ = C / Cs (where C is the lysozyme concentration, Cs is the lysozyme solubility). C can be estimated according to the initial concentration (20 mg/ml lysozyme, 30 mg/ml NaCl) and the change in the drop volume according to Equation (1). With the data for C and Cs, the nucleation rate I can be obtained according to Equations (2) - (5):

 C  Cs  I  Kn    Cs 

a

(6)

where kn and a are constants. As proposed by Saikumar et al.28, the nucleation rate can be estimated using the following equation:

 C  Cs  I  7.7110  2     Cs 

3

(7)

23



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Figure 10. The estimated supersaturation and nucleation rate under irradiation by different sound programs. (a) The variation of the supersaturation in the crystallization droplet and (b) estimated nucleation rate.

Figure 10 shows the estimated results of the supersaturation and the nucleation rate during the course of applying the sound programs. Both the supersaturation and nucleation rate were enhanced with irradiation by STW programs and 5000 Hz compared with the control. Comparison of the sound frequency program and fixed frequency sound showed that variation of the crystallization droplet volume had a border range with varied sound frequency programs compared to the fixed sound frequency program. Figure 7 show that the droplet volume increased 9.08% for the STW 2 program, 7.86% for the STW 1 program, and 4.35% for the 5000 Hz program. The supersaturation was higher in the varied frequency program (5.91 for the STW 2 program, 5.56 for the STW 1 program) compared with the fixed frequency program (4.79 for the 5000 Hz program). The nucleation rate was higher for the STW 1 program (7.30) and the STW 2 program (9.10) compared with the 5000 Hz program (4.21). We found that protein crystallization was promoted more significantly using audible sound that shifted from high to low frequency (136% increase) compared with that shifted from low to high frequency (61.9% increase). One possible explanation is that protein molecules were assembled faster using a high - to - low frequency 24


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program at the beginning stage. Figure 7 shows the increase in protein concentration was faster with a high – to - low frequency program (STW 2 program) compared with a low - to - high frequency program (STW 1 program) at the beginning stage (nucleation also happened at this time), so the nucleation rate was higher for the STW 2 program compared with the STW 1 program, as is shown in Figure 10.

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5. Conclusions We tested the effect of audible sound frequency programs on protein crystallization and verified that audible sound had a beneficial effect on protein crystallization. Among twelve different audible sound frequency programs, the STW 2 program (frequency varied linearly from 15000 Hz to 100 Hz in 12 h) showed the most effective promotion of protein crystallization. We also compared the effect of different sound programs (including fixed frequency sound 5000 Hz, STW 2 and STW 1) on protein crystallization and supersaturation and nucleation rates. STW 2 showed the most significant enhancement of protein crystallization, and it can also improve the crystal quality, so it can be proposed as an effective tool to promote protein crystallization.

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Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Summarization of the diffraction data of lysozyme crystals with and without sound. Summarization of the diffraction data of proteinase K crystals with and without sound.

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Acknowledgements This work was supported by National Natural Science Foundation of China (Grant Nos. 11202167 and U1632126), China Postdoctoral Science Foundation (Grant Nos. 2017M623248 and 2013T60890), Seed Foundation of Innovation and Creation for Graduate Students in Northwestern Polytechnical University (Grant No. Z2017238), Undergraduate Training Programs for Innovation and Entrepreneurship in China (Grant Nos. 201710699332 and 201810699351).

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AUTHOR INFORMATION Corresponding Author Chen-Yan Zhang: Phone: +86-29-88460543. Fax: +81-29-88460543. E-mail: [email protected] Da-Chuan Yin: Phone: +86-29-88460254. Fax: +81-29-88460254. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. C.Y.Z. and D.C.Y. designed research; J.L., M.Y.W., W.J.L., and N.J. performed research; C.Y.Z., M.Y.W., N.J., C.Q.Y., M.L.H., Y.L., X.Y.Y., and R.B.Z. analyzed data; and C.Y.Z., M.Y.W., and D.C.Y. wrote the paper. Funding Sources This work was supported by National Natural Science Foundation of China (Grant Nos. 11202167, U1632126) China Postdoctoral Science Foundation (Grant Nos. 2017M623248, 2013T60890) Seed Foundation of Innovation and Creation for Graduate Students in Northwestern Polytechnical University (Grant No. Z2017238) Undergraduate Training Programs for Innovation and Entrepreneurship in China (Grant Nos. 201710699332, 201810699351) Notes The authors declare no completing financial interest.

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References (1)Artusio, F.; Pisano, R. Surface-induced crystallization of pharmaceuticals and biopharmaceuticals: A review, Int. J. Pharm. 2018, 547, 190-208. (2)Minussi, R. C.; Pastore, G. M.; Durán, N. Potential applications of laccase in the food industry, Trends Food Sci. Technol. 2002, 13, 205-216. (3)Khurshid, S.; Saridakis, E.; Govada, L.; Chayen, N. E. Porous nucleating agents for protein crystallization, Nat. Protoc. 2014, 9, 1621-1633. (4)Mike, S.; Alexander, E. S. V. D. Role of clusters in nonclassical nucleation and growth of protein crystals, Proc. Natl. Acad. Sci. USA. 2014, 111, E546-E553. (5)Kundrot, C. E.; Judge, R. A.; Pusey, M., Snell, E. H. Microgravity and macromolecular crystallography, Cryst. Growth Des. 2001, 1, 87-99. (6)Chayen, N. E.; Saridakis E. Protein crystallization: from purified protein to diffraction-quality crystal, Nat. methods 2008, 5, 147-153. (7)Siseth, M. C.; Mayra, C. C.; Nicola, D.; Maurizio, P.; Adela, R. R.; Abel, M. Glucose isomerase polymorphs obtained using and ad hoc protein crystallization temperature device and growth cell applying and electric field, Cryst. Growth Des. 2016, 16, 1679-1686. (8)Robin, S.; Arne, M.; Daniela, B.; Karsten, D.; Markus, P.; Christian, B. Real-time observation of protein dense liquid cluster evolution during nucleation in protein crystallization, Cryst. Growth Des. 2017, 17, 954-958. (9)McPherson, A. Protein Crystallization, Methods Mol. Biol. 2017, 1607, 17-50. (10) Byington, M. C.; Safari, M. S.; Conrad, J. C.; Vekilov, P. G. Protein conformational flexibility enables the formation of dense liquid clusters: tests using solution shear, J. Phys. Chem. Lett. 2016, 7, 2339-2345. (11) Yang, X. Z.; Zhang, C. Y.; Wang, Q. J.; Guo, Y. Z.; Dong, C.; Yan, E. K.; Liu, W. J.; Zheng, X. W.; Yin, D. C. Utilization of cyclodextrins and its derivative particles as nucleants for protein crystallization, Cryst. Growth Des. 2017, 17, 6189-6200. (12) Yin, D. C. Protein crystallization in a magnetic field, Prog. Cryst. Growth Ch. 2015, 30


Page 31 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


61, 1-26. (13) Flores-Hernández, E.; Stojanoff, V.; Arreguín-Espinosa, R., Moreno, A.; Sánchez-Puig, N. An electrically assisted device for protein crystallization in a vapor-diffusion setup, J. Appl. Crystallogr. 2013, 46, 832-834. (14) Shah, U. V.; Amberg, C.; Diao Y., Yang, Z. Q.; Heng, J. Y.Y. Heterogeneous nucleants for crystallogenesis and bioseparation, Curr. Opin. Chem. Eng. 2015, 8, 69-75. (15) Zhang, C. Y.; Liu, Y.; Tian, X. H.; Liu, W. J.; Li, X. Y.; Yang, L. X.; Jiang, H. J.; Han, C.; Chen, K. A.; Yin, D. C. Effect of real-world sounds on protein crystallization, Int. J. Biol. Macromol. 2018, 112, 841-851. (16) Kakinouchi, K.; Adachi, H.; Matsumura, H.; Inoue, T.; Murakami, S.; Mori, Y.; Koga Y.; Takano, K.; Kanaya S. Effect of ultrasonic irradiation on protein crystallization, J. Cryst. Growth 2006, 292, 437-440. (17) Wakayama, N. I.; Yin, D. C.; Harata, K.; Kiyoshi, T.; Fujiwara, M.; Tanimoto, Y. Macromolecular crystallization in microgravity generated by a superconducting magnet, Ann Ny Acad Sci. 2006, 1077, 184-193. (18) Pérez, Y.; Eid, D.; Acosta, F.; Moreno, A. Electrochemically assisted protein crystallization of commercial cytochrome c without previous purification, Cryst. Growth Des. 2008, 8, 2493-2496. (19) Lu, Q. Q.; Zhang, B.; Tao, L.; Xu, L.; Chen, D.; Zhu, J.; Yin, D. C. Improving protein crystal quality via mechanical vibration, Cryst. Growth Des. 2016, 16, 4869-4876. (20) Zhang, C. Y.; Yin, D. C.; Lu, Q. Q.; Guo, Y. Z.; Guo, W. H.; Wang, X. K.; Li, H. S., Lu, H. M.; Ye, Y. J. Cycling temperature strategy: a method to improve the efficiency of crystallization condition screening of proteins, Cryst. Growth Des. 2008, 8, 4227-4232. (21) Zhang, C. Y.; Wang, Y.; Schubert, R.; Liu, Y.; Wang, M. Y.; Chen, D.; Guo, Y. Z.; Dong, C.; Lu, H. M., Liu, Y. M.; Wu, Z. Q.; Betzel, C.; Yin, D. C. The effect of 31



Page 32 of 33

audible sound on protein crystallization, Cryst. Growth Des. 2016, 16, 705-713. (22) Liu, Y. M.; Li, H. S.; Wu, Z. Q.; Chen, R. Q.; Lu, Q. Q.; Guo, Y. Z.; Zhang, C. Y.; Yin, D. C. Sensitivity of lysozyme crystallization to temperature variation, CrystEngComm 2016, 18, 1609-1617. (23) Minor, W.; Cymborowski, M.; Otwinowski, Z.; Chruszcz, M. HKL-3000: the integration of data reduction and structure solution--from diffraction images to an initial model in minutes, Acta Cryst. 2006, D62, 859-866. (24) Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.; Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G. W.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin A.; Wilson, K. S. Overview of the CCP4 suite and current developments, Acta Cryst. 2011, D67, 235-242. (25) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics, Acta Cryst. 2004, D60, 2126-2132. (26) Grell, L.; Parkin, C.; Slatest, L.; Craig P. A. A tool for simplifying molecular viewing in PyMOL, Biochem. Mol. Biol. Educ. 2006, 34, 402-407. (27) Forsythe, E. L.; Judge, R. A.; Pusey, M. L. Tetragonal chicken egg white lysozyme solubility in sodium chloride solutions, J. Chem. Eng. Data 1999, 44, 637-640. (28) Saikumar, M. V.; Glatz, C. E.; Larson, M. A. Lysozyme crystal growth and nucleation kinetics, J. Cryst. Growth 1998, 187, 277-288.

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For Table of Contents Use Only Protein crystallization irradiated by audible sound: the effect of varying sound frequency Chen-Yan Zhang†, *, Jie Liu†, Meng-Ying Wang†, Wen-Jing Liu, Nan Jia, Chang-Qing Yang, Ming-Liang Hu, Yi Liu, Xian-Yu Ye, Ren-Bin Zhou, 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 Shenzhen Research Institute of Northwestern Polytechnical University, Shenzhen 518057, PR China

The estimated supersaturation and nucleation rate under irradiation by different sound programs. Both the supersaturation and nucleation rate were enhanced with irradiation by STW programs and 5000 Hz compared with the control.


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Protein crystallization irradiated by audible sound: the effect of varying

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