Use of Protein Thin Film Organized by External Electric Field as a Template for Protein Crystallization.

The well-known difficulty to obtain high-quality protein crystals has motivated researchers to come up with new methods or modifications of established crystallization methods to stimulate the growth of good diffracting crystals. In the present work, a new approach, using a protein thin film organized by external electric field (EEF) as a template for protein crystal growth, is introduced. This method increased nucleation of hen egg white lysozyme (HEWL) in comparison with the classical vapor diffusion method, besides improving crystal morphology and size. X-ray diffraction analyses indicated improvements in crystal quality. When HEWL was crystallized at pH 6.2, in which this protein presents biological activity, the control crystal presented a poorly ordered crystalline structure and a low resolution cutoff at 3.42 Å, whereas the crystal grown with the EEF protein film revealed a high-resolution limit at 1.67 Å. These results suggest that protein films organized by EEF may improve protein crystals and their data quality.

Introduction

Protein crystallization is a process that requires specific conditions, involving a transition phase in which protein molecules are no longer soluble and form an organized core.[1] In this process, the limiting step is the nucleation,[2] which requires not only a supersaturated solution but also an ordered clustering between protein molecules to initiate crystal growth.[3] In some cases, it is possible to reduce the energy barrier for nucleation by the creation of a microenvironment that favors a higher local concentration of macromolecules.[4]

One of the methods used to reduce this energy barrier is heterogeneous nucleation.[2] In this method, nuclei are formed on suspended solid particles or surfaces in contact with the solution. These supports can facilitate the nucleation process by attracting the molecules electrostatically, hydrophobically, or through specific interactions, which enables it to occur in metastable conditions.[1]

There are several works in the literature that apply heterogeneous nucleation, using distinctive materials as support, such as polymeric films,[5] rat whiskers and horse hair,[4] bioactive gel–glass particles,[2] micromica and natural chlorite,[6] self-assembled monolayers,[7,8] and porous and nonporous microspheres.[9] In the majority of these works, an increase in the crystallization rate, an increase in the size and number of crystals formed, and the formation of crystals even under unfavorable conditions could be observed. However, different nucleating materials can affect differently the quality of the crystal formed because of incompatibilities between their crystal lattices. Occasionally, the binding of crystals to the nucleating agents can also hinder the harvest of single crystals for X-ray diffraction analyses. Thus, these methods still present limitations, especially with regard to the quality of the crystals formed.

An alternative to promote nucleation and to obtain quality crystals would be to use the protein itself as nucleating centers. In some techniques, small crystals of the protein are used to seed the crystallizing medium and to promote the growth of larger crystals.[10] Nevertheless, dissolution of the seed crystals in medium may happen, and there is the need to produce new seed crystals for each new experiment.

Another way of promoting nucleation and crystal growth is described in works with protein thin films.[11−14] In these studies, protein thin films are formed by the Langmuir–Blodgett (LB) technique, in which the protein solution is applied in a water–air interface followed by immediate submission to high pressure and then transferred to a glass slide. On this film, a small drop of protein solution is applied and the slide is reversed on the crystallization plate, as in the hanging drop vapor-diffusion method. With this technique, it was possible to obtain microcrystals of two proteins that, until then, had not been crystallized: bovine cytochrome P450scc and human kinase CKII.[12] In further works with lysozyme, thaumatin, ribonuclease A and proteinase K analyses revealed that the crystals generated with LB films were larger (about three times) than the crystals without films, using the same solutions, and presented the same resolution (1.6 Å) under X-ray diffraction analyses.[15]

At evaluating the effects of synchrotron radiation on crystals formed of LB films, they were found to be more organized, more stable under radiation and with better diffraction limits[13] (when compared with crystals obtained by the classical method), allowing the collection of higher quality data. Moreover, at comparing the electron density maps, structural differences between the models obtained with LB crystals and classic crystals were observed. Despite the unique characteristics of LB crystals, some difficulties persist when the method is used for determining the structures of some proteins, especially membrane proteins.

The application of an external electric field (EEF) in the crystallization solution has been extensively studied by several researchers.[16−21] The electric field would have two main effects on a supersaturated solution: molecular orientation and density fluctuation.[22] Density fluctuation occurs mainly due to the electromigration, which leads to an increase in local concentration,[23] whereas the molecular orientation is a result of the molecular polarization because the electric field creates an oriented dipole, leading to a preferential crystal orientation.[24] The effects of electromigration and molecular orientation caused by an electric field from an alternating current were observed in experiments with lysozyme in solution,[18] in which a concentration gradient was established and led to the formation of crystals near the cathode. The EEF application during crystal growth can also improve the crystal quality and extend the resolution, through the increase of the level of crystal internal organization, as verified in another study with lysozyme.[20]

The use of an EEF during protein thin film formation was first described in a study with GlnB from Herbaspirillum seropedicae.[25] When the electric field was applied, the protein molecules were clearly oriented, as observed by atomic force microscopy (AFM) images.[25] Thus, it is expected that the films formed by the electric field have the potential to act as nucleation centers, which is the starting point for the development of the method proposed in this work.

Therefore, this work attempts to combine the pH variation and the application of an EEF to form an organized protein thin film, which was used as a nucleation center for protein crystallization. The structure of proteins films were analyzed by AFM and Fourier transform infrared spectroscopy (FTIR), and the quality of the crystals was evaluated by X-ray diffraction. We showed that protein thin films organized by EEF can contribute as a nucleation center to protein crystallization.

Results and Discussion

Atomic Force Microscopy

The lysozyme films formed by drop deposition on siliconized glass slides created clusters, as previously reported in studies of lysozyme adsorption onto mica.[26−28] Kim et al. (2002) observed the formation of protein particles on mica surface with a height of 2.5 nm and lateral dimensions in the range 10–25 nm, which correspond to a monolayer of lysozyme with approximately five protein molecules per cluster.

In our study, we observed the formation of larger clusters, with sizes that vary according to the pH conditions. In sodium acetate buffer (pH 4.5), mainly supramolecular structures with (60 ± 20) nm (Figure a,b) were formed, whereas in phosphate buffer (pH 6.5), larger structures averaging (500 ± 120) nm (Figure d,e) were observed. Yet, in (2-(N-morpholino)ethanesulfonic acid) (MES) buffer (pH 6.2) and in (tris(hydroxymethyl)-aminomethane hydrochloride) (Tris-HCl) buffer (pH 8.0), clusters with (150 ± 50) and (250 ± 50) nm of lateral size (Figure a,b,d,e) were present. Because lysozyme presents an ellipsoidal form with dimensions of 3.0 × 3.0 × 4.5 nm,[29] the clusters observed can gather tens to hundreds of molecules. Although the protein concentration (1.43 μg·mL–1) used was sufficient for a monolayer deposition, the protein diffusion on the surface generated aggregates with one or more protein layers (4–40 nm high).

Figure 1

Topographical AFM images of protein films on siliconized glass slides: (a) lysozyme in sodium acetate buffer (50 mM, pH 4.5, NaCl 50 mM), with EEF application, (b) in the same solution without EEF application, and (c) only sodium acetate buffer solution, exposed to EEF; (d) lysozyme in phosphate buffer (50 mM, pH 6.5, NaCl 50 mM), with EEF application, (e) in same solution without EEF application, and (f) only phosphate buffer solution exposed to EEF. The direction of the applied EEF is from left to right.

Figure 2

Topographical AFM images of protein films on siliconized glass slides: (a) lysozyme in MES buffer (50 mM, pH 6.2, NaCl 50 mM), with EEF application, (b) in same solution without EEF application, and (c) only MES buffer solution exposed to EEF; (d) lysozyme in Tris-HCl buffer (50 mM, pH 8.0, NaCl 50 mM), with EEF application, (e) in same solution without EEF application, and (f) only Tris-HCl buffer solution exposed to EEF. The direction of the applied EEF is from left to right.

The application of an EEF during film formation strongly suggests a different distribution of supramolecular structures on the surface, when this is compared to the drop deposition films. As previously reported in a study with GlnB-Hs films,[25] the presence of the electric field improves the organization of structures on protein thin films. In sodium acetate buffer (pH 4.5) and in phosphate buffer (pH 6.5), a slight orientation of clusters is seen (Figure a,d), at 30° and 50° relative to the direction of electric field application, respectively. Nevertheless, in MES (pH 6.2) and Tris-HCl buffers (pH 8.0), a very clear orientation at around 130° and 110° (relative to EEF) can be observed (Figure a,d), respectively.

On the other hand, the drop deposition films presented a sparse distribution of supramolecular structures throughout the surface, leading to the formation of large clusters (Figure b,e) or having better area coverage (Figure b,e).

Once the buffer compounds form similar structures in drop deposition films,[25,30] AFM images have been taken from thin films formed by buffer solution exposed to EEF. These images showed no evidence of orientation and a random distribution of buffer compounds (Figures c,f and 2c,f), similar to the pattern seen in the drop deposition protein films. Thus, the orientation would be related to the presence of large polar molecules, such as proteins,[25] but not to buffer components. Despite this, the buffer composition directly interferes in the distribution profile of structures on protein thin films, when exposed to electric field, as can be seen in Figures a,d and 2a,d.

Fourier Transform Infrared Spectroscopy

The final averaged FTIR spectrum of lysozyme films for each buffer condition is shown in Figure S1. Although a spectrum range between 600 and 4000 cm–1 was used in the measurements, the amide I band, from 1600 to 1700 cm–1, was used to analyze the results. This band corresponds to the vibration mode mostly used in studies of protein secondary structure and originates mainly from C=O stretching of the amide group.[31] The Gaussian peak fitting procedure has been applied to the averaged FTIR spectra (OriginPro 8 software). To optimize the fit, eight Gaussian curves were used for spectral deconvolution. The quality of the fitting was evaluated on the basis of χ2 values (on the order of 10–7) and correlation coefficient values (≥0.999).

Hen egg white lysozyme structure is composed of six α-helices and three β-sheets connected by few flexible loops.[28] In solution, lysozyme exhibits an amide I maximum at 1654 cm–1, typical of a protein with predominant α-helix secondary structure, and minor absorptions at 1630 and 1673 cm–1 (corresponding to extended chains like in the β-sheet structure), 1641 cm–1 (unordered structures), and 1666 and 1682 cm–1 (turns and bends).[32] However, as hydrated film on an attenuated total reflectance (ATR) plate, lysozyme presents different frequency absorption, the maxima at 1646 and 1654 cm–1 (α-helix), 1630 cm–1 (β-sheet), and minor frequency absorptions at 1638 and 1662 cm–1 (random coil), and 1670 and 1678 cm–1 (β-turns).[33]

Comparing the FTIR spectra of lysozyme films formed by drop deposition (control) with the spectra of lysozyme films formed by EEF application, it was possible to observe a shift in the α-helix frequency, especially in the spectra of the protein in sodium acetate buffer and in Tris-HCl buffer (Figure S1). In Table , the deconvolved frequencies are summarized and it is possible to observe a shift from 1661 to 1657 cm–1, when the protein is in sodium acetate buffer, and from 1659 to 1656 cm–1, when the protein is in Tris-HCl buffer. This difference could be attributed to structural rearrangements of the protein.[31] The shift in the α-helix frequency was also observed in lysozyme Langmuir–Schaefer films when treated at high temperatures,[34] which is attributed to the stabilization of the helical structure upon removal of water molecules. The difference in the distribution of particles on the films may actually interfere with water adsorption, which also leads to a difference in the signal observed in the spectra of the control and the EEF films.

Table 1

Deconvolved Amide I Frequencies (cm–1) for Lysozyme Films

	lysozyme films formed with drop deposition (control)				lysozyme films formed with EEF
buffer solution	acetate (pH 4.5)	MES (pH 6.2)	phosphate (pH 6.5)	Tris-HCl (pH 8.0)	acetate (pH 4.5)	MES (pH 6.2)	phosphate (pH 6.5)	Tris-HCl (pH 8.0)
aggregated (1610–1628)a	1619	1621	1621	1619	1626	1625	1619	1621
β-sheet (1625–1640)a	1629	1639	1640	1633	1632	1633	1631	1631
random coil (1640–1648)a	1640			1644	1644	1643		1643
α-helix (1648–1660)a	1651	1650, 1656	1650, 1658	1651, 1659	1651, 1657	1650, 1657	1650	1650, 1656
310-helix (1660–1670)a	1661, 1666	1664	1670	1667	1664	1666	1661, 1669	1665
antiparallel β-sheet/turns (1675–1695)a	1684	1684	1684	1675, 1682	1677, 1691	1678, 1690	1680, 1694	1678, 1690

According to Jackson & Mantsch (1995).[31]

Concerning the β-sheet frequency, it was possible to verify a shift to a lower wavenumber in the spectra of the protein in MES and in phosphate buffers, from 1639 to 1633 cm–1 and from 1640 to 1631 cm–1, respectively. Furthermore, the region corresponding to antiparallel β-sheets or turns also presents a shift in the peaks. While absorptions at 1682–1684 cm–1 can be assigned in control films, the EEF films present absorptions at 1677–1680 and 1690–1694 cm–1. Once more, we can infer a structure reorganization of the protein after EEF application.

Crystallization under Different Conditions

The crystallization tests were performed at three different pH values: acid, employing sodium acetate (pH 4.5) as buffer, which is the most widely used pH for lysozyme crystallization; alkaline, using Tris-HCl (pH 8.0) as buffer; and the pH of lysozyme biological activity (pH 6.2), for which MES buffer was used.

In sodium acetate buffer, as expected, there were no difficulties for crystal appearance in a few days, even without the use of the protein film (Figure a,d). However, the use of the EEF film as a template allowed the formation of a higher number of crystals, clearly because of an increase in nucleation (Figure b,c). At conditions with lower salt concentration, in which there was precipitation in the control assays, the film allowed the growth of large and regular crystals. In some cases, the use of the film retarded the emergence of crystals; however, it contributed to the formation of larger and symmetrical crystals (Figure e,f), which can be important for improving data collected by X-ray diffraction.

Figure 3

Lysozyme crystals obtained by the classical vapor-diffusion method without the use of protein thin films (controls) (a,d,g,j,m,p) and using EEF protein thin films in phosphate buffer (b,e,h,k,n,q), in sodium acetate buffer (c,f), in MES buffer (i,l), and in Tris-HCl buffer (o,r). Reservoir solution: sodium acetate buffer 50 mM (pH 4.5) with 780 mM NaCl (a–c) and 900 mM NaCl (d–f); MES buffer 50 mM (pH 6.2) with 780 mM NaCl (g–i) and 1 M NaCl (j–l); Tris-HCl buffer 50 mM (pH 8.0) with 1 M NaCl (m–o) and 900 mM NaCl (p–r). Drop solution: 1:1, lysozyme 10 mg·mL–1 (a–c,g–i,m–o) and 20 mg·mL–1 (d–f,j–l,p–r). It is one representative experiment of three independent assays.

Nevertheless, at the pH condition in which the enzyme presents biological activity (MES buffer, pH 6.2), the control tests required more time (about 1 month) to crystallize, and moreover, only a few small crystals were formed (Figure g). Only at higher protein concentration, larger crystals could be obtained (Figure j). On the other hand, with the use of the EEF protein films, the time for crystal emergence was reduced to 1 week. In addition, nucleation was stimulated once again (Figure h,i), leading to the generation of several medium-size crystals. Although the growth of larger crystals took longer time (2–6 months), the method of EEF protein film produced regular crystals (Figure k,l).

Under alkaline conditions (Tris-HCl buffer, pH 8.0), a large number of small crystals appeared in the control tests, when lower protein concentration was used (Figure m). When a higher protein concentration was employed, either precipitation occurred (in some conditions) or crystals with an irregular shape were formed (Figure p). In contrast, the EEF protein films enabled the growth of larger and more symmetrical crystals (Figure q,r) in shorter time. Even at low protein concentrations, when the nucleation is favored, crystals of medium size were observed (Figure n,o).

The number of crystals obtained was significantly higher in the presence of protein thin film (p < 0.01) for acetate and MES buffer.

In addition, tests were performed with standard crystallization solutions (Hampton Research Crystal Screens, HR2-110 and HR2-112). Six solutions of different pH values from each crystallization kit were chosen. In some conditions, crystal formation was observed only when the EEF protein film was used as a template, namely, 100 mM imidazole (pH 6.5) with 1 M sodium acetate; 100 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (pH 7.5) with 100 mM NaCl and 1.6 M ammonium sulfate; and 100 mM sodium cacodylate (pH 6.5) with 200 mM magnesium acetate and 20% polyethylene glycol 8000. In other cases, such as in 100 mM MES (pH 6.5) with 200 mM ammonium sulfate and 30% polyethylene glycol monomethyl ether 5000, it was possible to observe an increase in the number of crystals formed.

Our results agree with studies in which the usage of LB protein thin films led to a positive influence in protein nucleation, with an acceleration of crystal growth rate, in comparison with the classical hanging drop method.[11,12] In addition, the crystals obtained with LB films are larger than those grown with the classical method,[11,15] which was also observed in our study.

Diffraction Quality Analyses of Lysozyme Crystals

The quality of lysozyme crystals grown under different conditions was analyzed by X-ray diffraction. For these experiments, crystals with good morphology and similar sizes for each pH condition were selected, such that more than one crystal from each situation was used. Table summarizes the diffraction data statistics for crystals grown with and without the use of EEF protein thin film as a template, obtained after data processing with XDS and XSCALE.

Table 2

X-ray Diffraction Data Statistics of Lysozyme Crystals Grown with and without EEF Protein Thin Filma

	lysozyme crystallized in sodium acetate buffer (pH 4.5)			lysozyme crystallized in MES buffer (pH 6.2)			lysozyme crystallized in Tris-HCl buffer (pH 8.0)
	control	crystal A1b	crystal A2c	control	crystal M1b	crystal M2c	control	crystal T1b	crystal T2c
space group	P43212	P43212	P43212	P43212	P43212	P43212	P43212	P43212	P43212
unit cell (Å)	a = 78.25, b = 78.25, c = 36.62	a = 78.25, b = 78.25, c = 36.62	a = 78.25, b = 78.25, c = 36.62	a = 77.29, b = 77.29, c = 36.71	a = 78.25, b = 78.25, c = 36.62	a = 78.25, b = 78.25, c = 36.62	a = 78.25, b = 78.25, c = 36.62	a = 78.25, b = 78.25, c = 36.62	a = 78.25, b = 78.25, c = 36.62
total reflections	95 608 (1604)	60 581 (5182)	82 883 (2966)	10 015 (1056)	73 719 (2557)	85 196 (3006)	93 631 (1691)	50 564 (1798)	83 139 (2985)
unique reflections	18 053 (874)	9074 (770)	13 402 (664)	1632 (180)	11 423 (540)	13 679 (687)	17 206 (623)	13 047 (587)	13 449 (643)
multiplicity	5.30 (1.84)	6.68 (6.73)	6.18 (4.47)	6.14 (5.87)	6.45 (4.74)	6.23 (4.38)	5.44 (2.71)	3.87 (3.06)	6.18 (4.64)
resolution range (Å)	78–1.51 (1.54–1.51)	78–1.92 (1.98–1.92)	78–1.68 (1.71–1.68)	78–3.42 (3.56–3.42)	78–1.77 (1.80–1.77)	78–1.67 (1.70–1.67)	78–1.52 (1.55–1.52)	78–1.67 (1.70–1.67)	78–1.67 (1.70–1.67)
completeness (%)	98.1 (85.2)	99.5 (99.6)	99.5 (99.3)	96.5 (98.4)	98.8 (98.7)	99.8 (98.7)	95.3 (62.2)	95.2 (84.3)	98.1 (92.4)
Rmeas (%)	5.9 (67.6)	17.5 (187.1)	10.4 (132.6)	41.0 (48.6)	11.7 (110.7)	8.2 (121.9)	4.5 (12.0)	3.2 (15.5)	7.9 (51.1)
I/σ(I)	15.00 (1.04)	8.02 (1.09)	11.91 (1.38)	2.98 (2.79)	10.67 (1.67)	15.09 (1.43)	24.28 (8.05)	29.85 (8.25)	14.40 (3.24)
CC1/2	99.9 (59.1)	99.4 (57.2)	99.8 (51.5)	94.6 (92.8)	99.7 (53.5)	99.9 (51.5)	99.9 (97.8)	99.9 (97.3)	99.7 (86.3)
mosaicity	0.29518	0.58000	0.30339	0.58234	0.25500	0.23221	0.27367	0.24687	0.28607

It represents the best data set collected for each condition tested. Three independent sets of data were collected for each condition.

Using as a template for crystal growth a film of lysozyme in crystallization buffer.

Using as a template for crystal growth a film of lysozyme in phosphate buffer.

In order to compare the quality of crystals, three concomitant criteria were used to determine the diffraction resolution limit: global completeness ≥95%, ⟨I/σ(I)⟩ ≥ 1.0 and CC1/2 ≥ 50% on the highest resolution shell. Recent studies have shown that, in some cases, there is useful information remaining even when CC1/2 falls to around 40–20% and ⟨I/σ(I)⟩ to around 1.5–0.5,[35] for model improvement. Despite being widely used for determining the resolution cutoff, Rmeas or Rmerge values should play no role to define the high-resolution limit because they can discard many useful data.[36,37]

The diffraction analyses indicated that crystals formed in sodium acetate buffer without protein film presented the highest resolution limit. This was already expected because it is the most widely used condition for lysozyme crystallization. Nevertheless, the EEF crystal obtained in phosphate buffer (crystal A2) also exhibited a good resolution limit. The lower resolution limit presented by the EEF crystal in sodium acetate buffer (crystal A1) could be related to the small conformational changes observed by FTIR analyses, which affect the crystalline structure and increase the crystal mosaicity, as can be seen in Table .

The lysozyme crystals obtained using LB films at the same buffer conditions were reported to show a high-resolution limit, at 1.57 Å, with ⟨I/σ(I)⟩ of 0.1 and completeness of 26.83%.[14] Despite the fact that resolution cut-off criteria for LB crystals were different from ours, we can say that our method propitiated also a high-resolution for EEF crystals in phosphate buffer, at 1.68 Å, with better diffraction quality ⟨I/σ(I)⟩ of 1.38 and completeness of 99.3%.

In alkaline conditions, where Tris-HCl buffer was used for crystallization, crystals obtained with EEF protein film demonstrated diffraction properties similar to control crystal, despite presenting slightly lower resolution limits. Interestingly, in this pH condition, the space group and the unit cell dimensions are exactly the same for the crystal in sodium acetate buffer. This fact may be related to the remarkable structural stability of lysozyme in different pH conditions.[38]

On the other hand, when MES buffer (pH condition of lysozyme biological activity) was used, EEF protein films led to a dramatic improvement in the diffraction properties of lysozyme crystals. The control crystal showed a less ordered crystalline structure, partly because of high mosaicity, resulting in lower diffraction power and resolution limit. Compared to the control, the resolution limit increased from 3.42 to 1.67 Å (with EEF film in phosphate buffer). In contrast to what occurred in sodium acetate buffer, the EEF protein film in MES and in phosphate buffer has apparently improved the crystalline structure of lysozyme crystals, allowing the better resolutions achieved by X-ray diffraction.

Conclusions

In this work, lysozyme crystallization was investigated using as nucleation center a protein thin film obtained with the application of an EEF. Different pH conditions were assayed, not only during the protein film preparation but also in the crystallization tests. Regarding to the lysozyme films, AFM images have suggested an orientation of structures when EEF was applied, especially in the ones where MES and Tris-HCl buffer were used. The FTIR spectra of lysozyme films demonstrated some small frequency shifts, indicating possible structural rearrangements of the protein in the EEF films.

Despite this, the organized structure of the EEF protein film has increased nucleation in all tested conditions, leading to the formation of a larger number of crystals and, sometimes, also contributing to the improvement of their size and/or morphology. Diffraction analyses of the crystals grown with EEF protein films revealed a significant improvement of the diffraction properties of the ones obtained at pH 6.2, which corresponds to the biological activity of lysozyme.

The results suggest that the EEF protein films could be particularly useful for the crystallization of proteins at lower initial concentration, thus using small protein amounts and not requiring crystallization kits to obtain crystals. Furthermore, this new approach opens possibilities to study a protein structure in its physiological condition, which may bring insights regarding its active structure. This method is easily performed in any laboratory.

Further work is necessary to understand the mechanisms of EEF protein thin film influence in protein crystallization and nucleation. Of remarkable importance, it would be to continue the investigation with other protein models, in order to verify if these achievements are reproducible.

Experimental Section

Protein Solutions

The classic protein crystallization model, with chicken egg white lysozyme (molecular weight 14 300 Da, cat. no. 62970, Sigma-Aldrich Ltd.), was used in our work. Lysozyme is a commercially available protein and has been used as a standard for the development of new methods for protein crystallization. The native protein solution was diluted to a final concentration of 1.43 μg·mL–1 (100 nM) in 50 mM sodium acetate (pH 4.5) and 50 mM NaCl; in 50 mM MES (pH 6.2) and 50 mM NaCl; in 50 mM sodium phosphate (pH 6.5) and 50 mM NaCl; and in 50 mM Tris-HCl (pH 8.0) and 50 mM NaCl, to be used for the protein thin film preparation. For crystallization experiments, the protein solution was diluted to 20 and 40 mg·mL–1 in purified water.

EEF Protein Thin Film Preparation

Each protein solution was dripped on siliconized cover slips (Hampton Research HR 3-231, 22 mm) inside channels (5 mm × 3 mm) made of silicon paste and conductive ink.[25] Each channel was filled with protein solution (20 μL), and then the EEF (300 V, 5 min) was applied using a conventional electrophoresis power supply. To determine the best conditions, the current was monitored as described in Ferreira et al. (2015). The same protein solutions were used as control, by simple drop deposition on siliconized cover slips. In addition, solutions prepared with only buffer components were dripped on channels and the same EEF was applied. The films were dried at room temperature.

AFM (Shimadzu SPM9500J3, Japan) was used to analyze the topology and phase of protein thin films, as well as of the buffer solutions and controls. The images were obtained in dynamic mode, with a scan rate of 1.3 Hz. A silicon cantilever with a spring constant of 36 N/m and a radius tip of 10 nm (Nanosensors) was used. The scan direction was aligned with the applied EEF. The measurements were performed at controlled temperature (22 °C) and humidity (40–50%). In order to eliminate noise and to correct the slope of piezoelectric, the images obtained were treated with Shimadzu software. Three random regions of 10–1 μm were chosen in each sample, in order to increase the statistical credibility of results and to describe accurately the structure of the film.

Fourier Transform Infrared Spectroscopy

In order to verify the structural integrity of the proteins in the film, after the EEF application, FTIR spectroscopy in ATR mode was used. Measurements were performed in a Bruker spectrometer (Vertex 70) with a ZnSe crystal, with the spectrum range between 600 and 4000 cm–1. All spectra corresponded to an average of 16 scans. The analyses were performed at controlled temperature (20 °C) and humidity (40%). Spectra of buffer solutions were also obtained, to be compared to the spectra of the protein in the same buffer solutions. After each measurement, the ZnSe crystal was washed with ethanol 70% and Milli-Q ultrapure water. The final spectra are the results of averages of samples in triplicates and measurements in duplicates. All spectra were deconvolved and analyzed with OriginPro 8 software, to identify the position of the main peaks in each condition.

Crystallization

The protein thin films organized with the EEF were used as templates for crystallization tests. The siliconized cover slips were placed on polyvinyl chloride rings, inside six-well tissue culture plates. With the purpose of determining the composition of reservoir solutions, a classic sitting drop test (without the use of protein thin film as a template) was performed. In this test, sodium acetate buffer 50 mM (pH 4.5) with NaCl (500 mM to 1000 mM) was used as the reservoir solution. For comparison, the same conditions were used in crystallization tests with EEF protein thin films. Because in both cases the best crystals were formed at NaCl 780 mM, 900 mM and 1000 mM, these salt concentrations were also employed in tests with other buffer solutions (at different pHs), especially Tris-HCl buffer (pH 8.0) and MES buffer (pH 6.2). In each condition, a classic sitting drop test, as well as assays using the protein film with lysozyme in the same reservoir buffer and with lysozyme in phosphate buffer (pH 6.5), was performed, as described by Pechkova & Nicolini[11] (2001). The ratio between the volumes of the crystallization drop and the reservoir solution was 1:1000. The crystallization experiments were carried out at 20 °C. All the experiments were repeated three times.

Data Collection and Processing

X-ray diffraction data were collected at MX2 beamline of the Brazilian National Synchrotron Laboratory (Campinas, Brazil[39]). The collection was performed at 100 K, the wavelength was 1.4586 Å, and the image exposure times for datasets were between 0.8 and 2.4 s. At least 200 well-diffracting images were obtained for each crystal using the PILATUS 2M detector. The crystal to detector distance was 0.100 m in all cases. The cryoprotector used was either 10% glycerol or 15% ethylene glycol, diluted in reservoir solution. The data were integrated, reduced, and scaled using XDS and XSCALE. A summary of the data collection statistics is given in Table . Three sets of data were collected for each experimental condition. Table shows the best set of data obtained for each condition.