Nonphotochemical Laser Induced Nucleation of Hen Egg White Lysozyme Crystals† In Sung Lee,# James M. B. Evans,§ Deniz Erdemir,#,⊥ Alfred Y. Lee,#,| Bruce A. Garetz,‡ and Allan S. Myerson*,#
CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 12 4255–4261
Department of Chemical and Biological Engineering, Illinois Institute of Technology, Chicago, Illinois 60616, Department of Chemical and Biological Sciences, Polytechnic UniVersity, Brooklyn, New York 11201, and NoVartis-MIT Center for Continuous Manufacturing, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 ReceiVed June 30, 2008; ReVised Manuscript ReceiVed September 30, 2008
ABSTRACT: Small droplets of supersaturated hen egg white lysozyme (HEWL) solution were exposed to intense linearly polarized laser pulses with different wavelengths, intensities, and pulse durations. Laser irradiation under some conditions significantly increased the number of droplets in which crystals were observed in a given time period, compared with nonirradiated samples. As a general rule, nonphotochemical laser induced nucleation (NPLIN) in lysozyme solutions was more effective with shorter aging time, 532nm wavelength, higher peak intensity, and shorter pulse duration. Bovine pancreatic trypsin (BPT) was also examined using NPLIN, showing the potential application of NPLIN to other proteins. Introduction Controlled protein crystallization is essential in structural biology because large high-quality crystals are required for structural determination which in turn provides information on the bioactivity of the proteins and aids in the design of drugs. Moreover, the use of protein crystals for pharmaceuticals, biosensors, biocatalysts and bioseparations has resulted in increasing interest in producing protein crystals with desired properties.1,2 Crystallization from supersaturated solutions involves nucleation and crystal growth. Nucleation, the initial step that entails the formation of a solid phase from a solution, is crucial in determining the size, shape, structure, and quality of the crystals obtained.3 Understanding and controlling the nucleation process is therefore vital in producing protein crystals of suitable quality for a variety of applications. Numerous attempts to control protein nucleation using techniques such as seeding,4 electric5,6 and magnetic fields,7 mineral surface,8 porous silicon,9 self-assembled monolayer,10 and poly-L-lysine modified substrate11 have been reported in the literature. However, obtaining suitable crystals is still challenging, as the underlying principles of the nucleation process in protein crystallization are not well understood.12 According to classical nucleation theory (CNT),13 molecules in a supersaturated solution aggregate into clusters that are assumed to be spherical and have a well-ordered crystalline structure. These clusters alternately grow and shrink until, by chance, a cluster reaches a critical size, after which the critical nucleus grows into a large detectable crystal.14 However, there is increasing evidence that the kinetics of protein nucleation does not agree with CNT predictions.15,16 Recently, dynamic and static light scattering17,18 and transmission electron micros* To whom correspondence should be addressed. E-mail:
[email protected]. † Part of the special issue (Vol 8, issue 12) on the 12th International Conference on the Crystallization of Biological Macromolecules, Cancun, Mexico, May 6-9, 2008. # Illinois Institute of Technology. ‡ Polytechnic University. § Massachusetts Institute of Technology. ⊥ Current address: Process Research and Development, Bristol-Myers Squibb Company, One Squibb Drive, New Brunswick, NJ 08903, USA. | Current address: Particle and Process Sciences and Engineering, Chemical Development, GlaxoSmithKline PLC, P.O. Box 1539, King of Prussia, PA 19406, USA.
copy19 experiments have suggested that there is a formation of an amorphous aggregation in the nucleation pathway from monomers to a mature crystal in protein systems. Furthermore, computer simulations have revealed that the high density fluctuations in protein solutions near the critical point lead to the formation of high density liquid protein phases followed by the reorganization of the protein molecules in order for macroscopic crystals to develop, thereby increasing the nucleation rate in the vicinity of the critical point for liquid-liquid separation.20 These simulations have been supported by density functional theory.21,22 It has been proposed that in supersaturated solutions, these dense liquid phases can occur not only near the critical point, but also far away from the liquid-liquid separation line, and that nucleation occurs after a structure fluctuation of protein molecules inside these dense liquid droplets, called two-step nucleation.23 A two-step nucleation mechanism has been suggested not only for proteins but also for small molecules.24-27 Recently, nonphotochemical laser-induced nucleation (NPLIN) has been demonstrated in the crystallization of small organic compounds such as urea and glycine.28-32 These experiments showed that exposure of intense nanosecond pulses of linearly or circularly polarized laser light into a supersaturated solution can significantly reduce the induction time and, in some systems, control the polymorphic form of crystals produced. Based on this, we have previously proposed that nucleation28-30 in these solutions of small molecules proceeds via a two-step mechanism. First, the solute molecules in a supersaturated solution aggregate to form disordered liquid-like clusters and second, the molecules inside liquid-like clusters rearrange into an ordered structure to produce the critical nucleus. We have proposed that the electric field generated by intense polarized laser pulses accelerates the reorganization of the liquid-like clusters into a crystalline structure by the optical Kerr effect, thereby reducing the nucleation induction time and, in some systems, controlling the polymorphic form of the crystal. Consequently, NPLIN is an effective method for studying and controlling nucleation. Other methods for controlling nucleation of proteins employing light irradiation have been demonstrated elsewhere. Veesler and co-workers have shown that the irradiation with light from a continuous Xe lamp of a metastable lysozyme solution
10.1021/cg800696u CCC: $40.75 2008 American Chemical Society Published on Web 11/06/2008
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promoted nucleation of lysozyme crystals.33,34 From the high correlation between the wavelengths for the efficiency of lightinduced nucleation and the absorption spectrum of lysozyme, they suggested a photochemical nucleation mechanism in which the absorption of light gives rise to lysozyme radicals, which are attracted to each other, thus enhancing the nucleation rate of lysozyme. Recently, high intensity femtosecond laser pulses35-37 and continuous wave (CW) laser light38,39 have been used for controlling crystallization of proteins as well as small molecules. The authors proposed that the irradiation of the metastable supersaturated solution with focused light triggers nucleation of crystals by the formation of critical-sized molecular clusters at the focal point via nonlinear effects such as shock wave generation or optical pressure driven by the high peak intensity femtosecond laser pulses or the continuous wave laser beam. In this paper, our aim is to explore light-induced protein nucleation in wavelength, intensity and pulse-duration regimes not covered by previous studies. To avoid the extreme nonlinear optical effects produced with focused femtosecond pulses, we have restricted our laser sources to nanosecond and picosecond pulse durations, and have not used focused laser beams, instead attempting to uniformly illuminate the whole volume of the solution droplet. We have also avoided ultraviolet wavelengths in order to reduce the chances of inducing photochemistry. However, it is well-known that the absorption of light by water is greatly dependent on the wavelength,40 and solution heating is strongly related to pulse duration and pulse repetition rate.36 In this work, NPLIN was carried out using three different laser sources with different wavelengths, intensities, and pulse durations in order to compare their effectiveness in controlling nucleation of a model protein, hen egg white lysozyme (HEWL) and to provide insight into the mechanism of nucleation. Bovine pancreatic trypsin (BPT) was also examined to investigate the application of NPLIN to other proteins. Experimental Procedures Lysozyme Crystallization. HEWL from Roche (Cat. No. 10 837 059 001) was used without further purification. Three lysozyme stock solutions were prepared by dissolving 400, 600, and 800 mg of HEWL in 10 mL of 0.1 M acetate aqueous buffer solution of pH 4.35, followed by filtration with a 0.22 µm syringe filter (Millipore Millex-GV). 500 µL of a 6% NaCl in 0.1 M acetate aqueous buffer solution of pH 4.35 was added slowly into a 500 µL sample of the lysozyme stock solution. Two µL of the prepared solution was then placed into a glass microbeaker (2 mL) filled with a 1 mL paraffin oil (Hampton Research), which prevents evaporation of water from the droplet.41 The inside surface of the bottom of microbeaker was siliconized with Rain X, purchased from Hampton Research, to reduce the contact surface area of the droplets on the bottom of microbeaker and to ensure that the droplets are as spherical as possible which should reduce the potential effect of the surface on the nucleation of the protein.42,43 Bovine Pancreas Trypsin Crystallizaton. Bovine pancreas trypsin (BPT) was purchased from Sigma-Aldrich and used without further purification. BPT stock solution was prepared by dissolving 400 mg of BPT in 10 mL of 10 mM calcium chloride, 10 mg/mL benzamidine hydrochloride, and 25 mM Hepes solution of pH 7.0, followed by filtration through a 0.22 µm syringe filter (Millipore Millex-GV). A 0.2 M sodium acetate trihydrate and 30% w/v polyethylene glycol 8000 in 0.1 M sodium cacodylate buffer solution of pH 6.5 was used as the precipitant and reservoir solution. A hanging drop vapor diffusion experiment was conducted using a 96-micro well quartz plate of which each well was filled with 200 µL of reservoir solution. A 2 µL droplet of stock solution was suspended with micropipet in the fixed positions of the siliconized microscope slide, followed by the addition of 2 µL precipitant solution into each stock solution droplet. The microscope slide on which droplets was placed was inverted to set each droplet into each well. High vacuum grease from Hampton was used to seal the microslide to the quartz plate.
Lee et al.
Figure 1. The schematic diagram of the experimental apparatus for the generation of linearly polarized intense laser beam. Laser Beam Generation. The schematic diagram for the generation of the intense linearly polarized laser beam is shown in Figure 1. Annular nanosecond near-infrared (NIR) and green laser light beams were generated by a Quantel Brilliant Q-switched Nd:YAG laser (with a second harmonic generator for green light), producing a 20 pulse/s train of 5 ns laser pulses at 1064 nm and 4 ns at 532 nm. The annular laser beam goes through a 3.5 mm diameter circular bronze aperture to select a small circular portion of the beam with roughly constant intensity, followed by a quartz zero-order half-wave retardation plate and finally a Glan-Thompson prism polarizer to produce the linearly polarized laser light beam either at 1064 or 532 nm. Annular picosecond green light was created by a Quantel YG501 laser, with 100 ps duration and 30 Hz repetition rate at 532 nm. The 3.5 mm annular laser light beam from the picosecond green laser is linearly polarized with the same procedure as described above for the nanosecond laser light beam. To increase the energy per unit area, the spot size of beam is reduced from 3.5 mm to 2.0 mm in diameter with a beam reducer. The laser power was measured with a coherent LM30-V power meter, designed for high intensity used at both 1064 and 532 nm. The pulsed laser beam was collinear with a low-power cw HeNe laser in order to aid the accurate alignment of the pulsed laser beam. Statistical Analysis of the Data. Our working hypothesis is that irradiated samples exhibit a crystallization behavior that is measurably different from nonirradiated samples, and that this difference is statistically significant. Protein solution droplets are divided into two groups: those that are exposed to laser pulses 30-60 min after solution preparation and those that are not exposed (control group). Twentyfour hours after solution preparation, all droplets are viewed with an optical microscope to determine if any protein crystals have formed. A measurement X is the percentage of a given group of droplets in which crystals have formed. The hypothesis test was employed, to determine if there is a statistical significance in the crystallization efficiency for irradiated and control groups with the null hypotheses; H0: XI ) XC;
Z0 )
XI - XC
√
x )
(
x(1 - x)
1 1 + SI SC
)
XISI + XC SC SI + SC
where SI and SC are the number of total samples of an irradiated group and a control group, respectively. With Z0 calculated and Φ(|Z0|) given by44
Φ(|Z0|) )
∫-∞Z √ 1 0
e-u ⁄2 du 2
2π
the p-value can be acquired by using:
p-value ) 2[1 - Φ(|Z0|)] If the p-value is less than R, it can be said that there is a statistically significant difference between the irradiated and control group in crystallization efficiency with 100(1 - R)% confidence. However if the p-value is greater than R, then there is no statistically significant difference at the 100(1 - R)% confidence level.44
Results and Discussion A series of NPLIN experiments with lysozyme were conducted by the microbatch method in which paraffin oil was used
Laser Induced Nucleation of HEWL Crystals
Figure 2. (a) Microbatch method and direction of laser beam, (b) hanging drop method with 96 quartz cells and direction of laser beam.
to prevent the evaporation of water, therefore maintaining constant supersaturation levels.41 A 60 mg/mL lysozyme stock solution of 500 µL was mixed with the same amount of precipitant solution. Immediately, each 2 µL droplet of 30 mg/ mL lysozyme in 3% NaCl, 0.1 M sodium acetate pH 4.35 was placed into the microbeaker filled with paraffin oil. The small droplet having a diameter of approximately 1.5 mm was exposed from the bottom to a few pulses or several seconds of continuous exposure to the intense, pulsed linearly polarized laser beam of diameter (3.5 mm or 2 mm) from two different types of light sources (Figure 2A). This was done at 30-60 min after mixing of the stock and precipitant solutions. The sealed microbeakers were kept at a constant temperature of 23 ( 2 °C. Table 1 shows the percentage of droplets in which crystallization occurred in 24 h after the irradiation. Irradiation with the high intensity nanosecond NIR laser with peak intensity of 0.125 GW/cm2 and nanosecond green laser with the peak intensities of 0.0032 and 0.0063 GW/cm2 appear to have had no effect on the nucleation compared to the nonirradiated control samples. In contrast, 10 s of continuous exposure to the intense, pulsed linearly polarized laser beam employing both the picosecond green laser, with peak intensities of 0.22-0.25 GW/cm2, and the nanosecond green laser with the peak intensities of 0.0095 and 0.026 GW/cm2 dramatically increased the percentage of droplets in which crystals were observed within 24 h of irradiation. Statistical analysis of the irradiated and control samples (Table 1) show that for both the picosecond green laser and nanosecond green laser with peak intensities of 0.0095 and 0.026 GW/cm2, a statistically significant effect was observed with p-values of less than 0.005. On the other hand, for the nanosecond green laser with peak intensities of between 0.058 and 0.068, the p-value of 0.08 indicates that the null hypothesis (H0: XI ) XC) cannot be rejected at the 95% confidence level, but will be rejected with 92% confidence. Analysis of the samples that were exposed to 10 s of high intensity nanosecond NIR beam does not show any statistically significant difference when compared to the control samples, with p-value of 0.67. We hypothesize that the failure of the NIR laser in these experiments is due to the absorption of significant quantities of energy at this wavelength (water absorption coefficient at 1064 nm is approximately over 0.61 cm-1),40 since the absorption of this energy in the 2 µL droplets equates to a rise in temperature of approximately 7 to 8 °C. This increase considerably reduces the supersaturation level of lysozyme solution45 and hence the number of liquid-like clusters over the critical size, which significantly decreases the efficiency of NPLIN. The failure of the NIR laser in inducing nucleation was unexpected as previous work has suggested that this type of irradiation was effective in inducing the nucleation of lysozyme.46 However, in previous work, laser light was focused
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on a small portion of a sample with larger volume (5-500 µL) such that the temperature rise of a solution would not be as significant as in our current work. In contrast, the absorption of green light by water is known to be small when compared to that of NIR.40 Therefore, the energy absorbed by water for the 10 s exposure to green light will have a very small impact on the temperature and thus would not reduce the supersaturation. High peak intensities of the picosecond green laser could potentially increase the efficiency of NPLIN for the nucleation of lysozyme, as such a result was reported for urea.31 This continued increase in nucleation efficiency with peak intensity was not observed employing the nanosecond green laser. In this case, the efficiency of NPLIN was the highest with the peak intensity of 0.026 GW/cm2, but for higher peak intensities between 0.058 and 0.068 GW/cm2 a lower efficiency of NPLIN was observed. This finding is unexpected when compared with the previous results which show higher NPLIN efficiency with higher peak intensity.31 Instead, the results suggest that the highest intensity nanosecond laser pulses might be increasing the temperature of small solution droplets due to the nonlinear effects such as multiphoton absorption. From earlier studies on the crystallization of urea and glycine with NPLIN, Garetz et al.28-30 proposed that the nucleation of urea and glycine proceeds in a two-step manner and that NPLIN may dramatically reduce the time taken in aligning molecules into an ordered crystalline structure by optical Kerr effect. However, it is unlikely that rigid large protein molecules are aligned in the direction of the electric field by irradiation with polarized laser light having short pulse duration such as 100 ps, owing to the long rotational relaxation times of proteins. For instance, let τr and τp be the protein rotational relaxation time and the laser pulse duration, respectively. Provided that OMAX is the maximum amount of orientation that develops for an optical electric field applied at time t ) 0, the actual orientation, O, that develops after a time, t, is given by
O ) OMAX[1 - exp(-t ⁄ τr)] The fractional orientation, F, that develops is
F ) O ⁄ OMAX ) 1 - exp(-t ⁄ τr) The fractional orientation that develops from a laser pulse of duration τp is obtained by setting t ) τp:
F ) 1 - exp(-τp ⁄ τr) If the pulse duration is much shorter that the relaxation time, τp , τr, then exp (-x) ≈ 1-x, where x ) τp/τr, yielding the result:
F ) τp ⁄ τr The rotational diffusion coefficient, Drot, of lysozyme in the solution with 30 mg/mL lysozyme and 3% NaCl was estimated to be less than 1.7 × 107 s-1.47 Using the correlation between the rotational diffusion coefficient and a relaxation time of lysozyme given by48,49
τr ) (6Drot)-1 the relaxation time of lysozyme is expected to be greater than 10 ns. Therefore, for lysozyme with a relaxation time of 10 ns and a laser a pulse duration 100 ps, F is 0.01. In other words, the amount of orientation that develops is one hundredth of what would develop if the pulse duration were much longer that the relaxation time. On the other hand, it is well recognized that a slight change in the degree of anisotropic interaction between protein mol-
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Table 1. Percentage of Droplets in Which HEWL Crystals Were Observed within 24 h at Various Conditions laser
nanosecond NIR laser
picosecond green laser
wavelength (nm) average intensity (mW/mm2) pulse duration pulse repetition rate (Hz) peak intensity (GW/cm2) exposure time (s) no. of laser pulses spot size (diameter, mm) diameter of droplet (mm) accumulated energy (mJ) percentage of droplets nucleated (%) no. of total samples p-value
1064 125 5 ns 20 0.125 10 200 3.5 1.5 2209 20.0 30 0.67
532 6.7-7.6 100 ps 30 0.223-0.257 10 300 3.5 1.5 118-134 83.3 60
