REVIEW OF SCIENTIFIC INSTRUMENTS 86, 015112 (2015)
Method and apparatus for characterization of electric field-induced aggregation in pre-crystalline protein solutions Takashi Wakamatsua) Department of Electrical and Electronic System Engineering, National Institute of Technology, Ibaraki College, Hitachinaka, Ibaraki 312-8508, Japan
(Received 28 September 2014; accepted 9 January 2015; published online 29 January 2015) The article presents a method and an apparatus for the characterization of protein aggregation under an applied internal electric field. The method is based on a forward light scattering technique that is highly sensitive to aggregates in pre-crystalline protein solutions. Transparent conductive films are used as electrodes for a planar thin sample cell, which enables precise measurement of the forward light scattering at small angles through the electrodes. Evaluation of the protein aggregation under applied electric fields was demonstrated for a model lysozyme protein. In situ measurements of crystallizing lysozyme solutions under a low applied voltage revealed that the forward static light scattering profiles changed with time into power law profiles. This indicates the formation of lysozyme fractal clusters under applied electric fields in the pre-crystalline state. The method and the apparatus presented here can sensitively evaluate the promotion process in protein crystallization under an applied electric field. C 2015 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4906328]
I. INTRODUCTION
In protein crystallography, the lack of an efficient production method for protein crystals of suitable quality for X-ray or neutron diffraction is a critical problem.1–4 Protein crystallization remains a trial and error procedure, performed by varying the physical and chemical parameters in a protein solution, including the concentrations of both protein and precipitant, and use of different precipitants, pH values, and crystallizing solution temperatures. Additionally, the protein crystallization process can take a long time to complete, from several days to even months. Therefore, a crystallization promotion technique and a sensitive analysis method for the proteins in the pre-crystalline state are required for improvement over the current crystallization procedure. Several methods to promote protein crystallization have been reported and demonstrated, which are based on application of external stimuli to metastable protein solutions, such as magnetic fields,5–7 ultra-short pulsed lasers,8,9 and electric fields.10–16 Among these methods, the electric field application technique is advantageous because it can be performed using simple and low-cost equipment. However, external electric field-induced protein crystallization typically requires the application of high voltages up to several kilovolts because the protein solutions are electrically isolated from the electrodes.10–12 This prevents the miniaturization of the crystallization sample cells. In contrast, our work17 has enabled the development of a small transparent cell for protein crystallization using transparent conductive electrodes composed of indium-tin-oxide (ITO) thin films that apply an internal electric field to the protein solutions directly. We
a)Author to whom correspondence should be addressed. Electronic mail:
[email protected] 0034-6748/2015/86(1)/015112/8/$30.00
demonstrated crystallization promotion for a typical lysozyme protein under a very low voltage of 80% in the visible range. The cells used for the FLS measurements had the same structures as the electric field-induced crystallization cells described earlier. The cell structure results in a light path length of 1.0 mm through the solution. The thin structures and the transparency of the cells enable precise measurement of the FLS at small angles from the protein sample solution through the electrodes under applied electric fields. Figure 2 shows a schematic of the apparatus used for the forward-SLS measurements, which was constructed in-house.
FIG. 2. Schematic diagram of the apparatus used to evaluate pre-crystalline protein aggregation under an applied electric field, based on in situ FLS measurements.
015112-4
Takashi Wakamatsu
The sample cell containing the protein solution was mounted on the θ rotation stage of a θ-2θ coupling stage with a common axis (Sigma Koki SGSP-H120YAW). The θ and 2θ stages were controlled using a controller (Sigma Koki SHOT-202) and a personal computer. The ITO-film-coated glass plate cell containing the sample solution was attached to a hemi-cylindrical BK-7 glass prism (10 mm radius and 30 mm high), which was fixed in a prism holder on the θ stage using immersion oil (Olympus IMMOIL) with the same refractive index as that of the glass. The ITO film electrodes of the sample cell were attached to fine metal wires using a silver particle paste and were then connected to the multifunction synthesizer or the LCR meter through these wires. The voltage waves that were applied to the cells were monitored using the storage oscilloscope. A diode-pumped solid state laser (Cobolt Blues, λ = 473 nm, 25 mW) was used for the SLS measurements. The laser generated a narrow light beam with a diameter of approximately 0.7 mm and a spread angle of
Method and apparatus for characterization of electric field-induced aggregation in pre-crystalline protein solutions Takashi Wakamatsua) Department of Electrical and Electronic System Engineering, National Institute of Technology, Ibaraki College, Hitachinaka, Ibaraki 312-8508, Japan
(Received 28 September 2014; accepted 9 January 2015; published online 29 January 2015) The article presents a method and an apparatus for the characterization of protein aggregation under an applied internal electric field. The method is based on a forward light scattering technique that is highly sensitive to aggregates in pre-crystalline protein solutions. Transparent conductive films are used as electrodes for a planar thin sample cell, which enables precise measurement of the forward light scattering at small angles through the electrodes. Evaluation of the protein aggregation under applied electric fields was demonstrated for a model lysozyme protein. In situ measurements of crystallizing lysozyme solutions under a low applied voltage revealed that the forward static light scattering profiles changed with time into power law profiles. This indicates the formation of lysozyme fractal clusters under applied electric fields in the pre-crystalline state. The method and the apparatus presented here can sensitively evaluate the promotion process in protein crystallization under an applied electric field. C 2015 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4906328]
I. INTRODUCTION
In protein crystallography, the lack of an efficient production method for protein crystals of suitable quality for X-ray or neutron diffraction is a critical problem.1–4 Protein crystallization remains a trial and error procedure, performed by varying the physical and chemical parameters in a protein solution, including the concentrations of both protein and precipitant, and use of different precipitants, pH values, and crystallizing solution temperatures. Additionally, the protein crystallization process can take a long time to complete, from several days to even months. Therefore, a crystallization promotion technique and a sensitive analysis method for the proteins in the pre-crystalline state are required for improvement over the current crystallization procedure. Several methods to promote protein crystallization have been reported and demonstrated, which are based on application of external stimuli to metastable protein solutions, such as magnetic fields,5–7 ultra-short pulsed lasers,8,9 and electric fields.10–16 Among these methods, the electric field application technique is advantageous because it can be performed using simple and low-cost equipment. However, external electric field-induced protein crystallization typically requires the application of high voltages up to several kilovolts because the protein solutions are electrically isolated from the electrodes.10–12 This prevents the miniaturization of the crystallization sample cells. In contrast, our work17 has enabled the development of a small transparent cell for protein crystallization using transparent conductive electrodes composed of indium-tin-oxide (ITO) thin films that apply an internal electric field to the protein solutions directly. We
a)Author to whom correspondence should be addressed. Electronic mail:
[email protected] 0034-6748/2015/86(1)/015112/8/$30.00
demonstrated crystallization promotion for a typical lysozyme protein under a very low voltage of 80% in the visible range. The cells used for the FLS measurements had the same structures as the electric field-induced crystallization cells described earlier. The cell structure results in a light path length of 1.0 mm through the solution. The thin structures and the transparency of the cells enable precise measurement of the FLS at small angles from the protein sample solution through the electrodes under applied electric fields. Figure 2 shows a schematic of the apparatus used for the forward-SLS measurements, which was constructed in-house.
FIG. 2. Schematic diagram of the apparatus used to evaluate pre-crystalline protein aggregation under an applied electric field, based on in situ FLS measurements.
015112-4
Takashi Wakamatsu
The sample cell containing the protein solution was mounted on the θ rotation stage of a θ-2θ coupling stage with a common axis (Sigma Koki SGSP-H120YAW). The θ and 2θ stages were controlled using a controller (Sigma Koki SHOT-202) and a personal computer. The ITO-film-coated glass plate cell containing the sample solution was attached to a hemi-cylindrical BK-7 glass prism (10 mm radius and 30 mm high), which was fixed in a prism holder on the θ stage using immersion oil (Olympus IMMOIL) with the same refractive index as that of the glass. The ITO film electrodes of the sample cell were attached to fine metal wires using a silver particle paste and were then connected to the multifunction synthesizer or the LCR meter through these wires. The voltage waves that were applied to the cells were monitored using the storage oscilloscope. A diode-pumped solid state laser (Cobolt Blues, λ = 473 nm, 25 mW) was used for the SLS measurements. The laser generated a narrow light beam with a diameter of approximately 0.7 mm and a spread angle of
