CRYSTAL GROWTH & DESIGN
Virus and Protein Crystallization under Hypergravity
2008 VOL. 8, NO. 8 2964–2969
Bernard Lorber* Architecture et Re´actiVite´ de l’ARN, UniVersite´ Louis Pasteur de Strasbourg, CNRS, IBMC, 15 rue Rene´ Descartes, 67084 Strasbourg, France ReceiVed January 19, 2008; ReVised Manuscript ReceiVed March 3, 2008
ABSTRACT: One small RNA plant virus and three monomeric and small molecular mass proteins (Mr ) 14500-22200) were crystallized in a centrifuge at gravity levels between 1000 and 22000 g under conditions where controls at unit gravity are soluble. Solubility measurements indicate that all crystals have grown in solutions that are metastable, i.e., insufficiently supersaturated to nucleate under normal conditions. Upon centrifugation, particle sedimentation generates a concentration gradient. At highest local concentration, supersaturation is shifted beyond supersolubility and the critical driving force required for nucleation is overcome. A simple procedure has been implemented for sample microvolumes. The habit of protein crystals grown under hypergravity diverges from that of reference crystals but their unit cells are unchanged. Centrifugation is applicable to the crystallization of dilute samples of biological particles with a wide range of sizes, from large viruses to small proteins. It provides a means to control the onset of nucleation and in some cases to accelerate crystal growth. 1. Introduction The crystallization of biological macromolecules and assemblies is influenced by numerous variables.1,2 Frequently, biochemical purity and homogeneity are responsible for batch to batch variations. The effects of physical chemical properties of the solvent including pH, ionic strength, neutral moment, or dielectric constant are more predictable. Temperature and pressure affect crystal nucleation and growth as in the case of inorganic or small organic compounds. Electric or magnetic fields may also have measurable influences on macromolecular crystallization.3,4 Studies of the influence of gravitational fields have unveiled interesting advantages. The reduced convection existing under microgravity has been demonstrated to result in more order in the crystal lattice.5,6 At the other extreme, hypergravity is not at all incompatible with the growth of good-quality crystals. Indeed, birefringent hexagonal beef liver catalase crystals prepared in a centrifuge exhibit normal X-ray diffraction patterns. Also, irregularly shaped crystals of a fungal catalase (Mr 240 000) are suitable for structure determination at 2 Å resolution.7–9 On the basis of these and other experimental data on catalases, it has been postulated that dilute solutions can give rise to ordered crystals when macromolecules are confined in a small fraction of the volume and that mass transport by sedimentation and diffusion interferes with nucleation and growth kinetics.10 In the course of the study of the crystallogenesis of an icosahedral virus, experimental phase diagrams have suggested that the viral particles should crystallize at very low precipitant concentration.11 Centrifugation appeared to be a straightforward and rapid way to increase the virus concentration while keeping all other parameters constant. Because this approach has immediately produced crystals, investigations have been pursued to see if it is also applicable to proteins with molecular masses of less than a tenth of that of catalase. This paper reports how centrifugation increases the local macromolecular concentration and triggers crystal nucleation * Corresponding author. Phone: 333 8841 7008. Fax: 333 8860 2218. E-mail:
[email protected].
at low precipitant concentration. A simple procedure implemented for microvolumes of dilute samples was validated with pure batches of a RNA plant virus (Mr 8.8 106) and of three monomeric proteins with Mr between 14500 and 22200. In all cases crystals grew in a limited time interval in solutions that do not crystallize under unit gravity. Solubility measurements were used to compare macromolecular supersaturation in centrifuged assays and in controls. Further, crystals were characterized by X-ray diffraction and their growth rates estimated. The results are compared to those of catalase, apoferritin (Mr 450 000), and ferritin (Mr 950 000) and discussed in the context of the application of hypergravity for a better control of protein crystal nucleation and growth. 2. Experimental Section 2.1. Chemicals, Virus, and Proteins. Polymethyl-3,3,3-trifluoropropylsiloxane (Cat. No. PS181, avg. Mr ) 2320, density 1.25 mg mL-1, kinematic viscosity 300 cSt, refractive index 1.381) was purchased from ABCR-Roth (Karlsruhe, Germany). Concentrated precipitant solutions (polyethylene glycol PEG-8000 and ammonium sulfate with Cat. Nos. HR2-535 and HR2-541, respectively) and fluorocarbon Fluorinert FC-70 (Cat. No. HR2-797, avg. Mr 820, density 1.94 mg mL-1, kinematic viscosity 12 cSt, refractive index 1.303) were purchased from Hampton Research (Aliso Viejo, CA). N-[2-acetamido]2-iminodiacetic acid (ADA) (Cat. No A-9883) was from Sigma. Other chemicals were of ACS grade. Tomato bushy stunt virus (TBSV) propagated and purified as described was spectrophotometrically pure.11 Six-times crystallized hen lysozyme (Cat. No 100940, lot E94Z05, Seikagaku Corp., Japan), crystallized turkey lysozyme (Cat. No. L-62255, lot 64H7230, Sigma), and sweet-tasting thaumatin (Cat. No. T-7638, lot 108F00299, Sigma) were very pure according to gel electrophoresis. The Mr and extinction coefficients of model particles are listed in Table 1. 2.2. Crystallization Assays. The compositions of solutions that lead to the crystallization of the particles used in this study are well-known.11–14 Crystallization in hypergravity was performed on 100 µL solution deposited on 20 µL silicone oil or fluorocarbon. For the duration of the centrifugation, controls were kept at the same temperature in vibration-free incubators (Model IPP200, Memmert, Germany). Assays were examined with a binocular microscope at a 20-50-fold magnification. 2.3. Centrifugation. Crystallizations were performed in a Sorvall Discovery M150SE microultracentrifuge (Hitachi, Japan). Because of the small size and mass rotors, low-volume armored chamber, temperature adjustment by Peltier effect, and quiet vacuum pumping, this centrifuge operates quasi without vibration. Two fixed-angle
10.1021/cg800073t CCC: $40.75 2008 American Chemical Society Published on Web 06/19/2008
Virus and Protein Crystallization under Hypergravity
Crystal Growth & Design, Vol. 8, No. 8, 2008 2965
Table 1. Structural Properties of Model Particlesa biological particle
dry Mr
hen lysozyme turkey lysozyme thaumatin TBSV
1.43 × 10 1.42 × 104 2.22 × 104 8.8 × 106 4
Vj (mL g-1)
ε (M-1 cm-1)
Sw (10-13 s)
D0 (10-7 cm2 s-1)
dh (nm)
0.73 0.73 0.72 0.71
3.8 × 10 3.9 × 104 2.8 × 104 4.4 × 107
1.9 1.9 2.2 132
12 12 9.4 1.1
3.4 3.4 4.6 37
4
a Protein relative molecular masses (Mr) and partial specific volumes (Vj) are computed from amino acid composition. Those for TBSV and mean sedimentation velocities (Sw) in water are taken from literature. Molar extinction coefficients () are for a wavelength of 280 nm in the case of the proteins and 260 nm in that of TBSV. The translational diffusion coefficient (D0) extrapolated to zero particle concentration in the crystallization buffer was determined by dynamic light scattering. The hydrodynamic diameter (dh) is derived from D0 assuming that particles are spheres. Sw, D0 and dh are given for a temperature of 20°C.
rotors have been used. The S100-AT3 titanium rotor (angle 30°, K factor 7.0, max. 100 000 rpm, min. 330 000 g, max. 435 000 g, mass 0.52 kg) accommodates 20 polycarbonate tubes (o.d. 7 mm, length 20 mm, wall thickness 1 mm, and volume 230 µL) with sampleto-axis distances of max. 3.90 cm, avg. 3.43 cm, and min. 2.96 cm. The sample tubes were sealed with two layers of Parafilm foil (Péchiney Plastic Packaging, Menasha, WI, USA). The S45A aluminum rotor (angle 45°, K factor 66.6, max 45000 rpm, min. 73 000 g, max. 124 000 g, mass 1.0 kg) accommodates 12 standard 1.5 mL microcentrifuge tubes with sample-to-axis distances of max. 5.52 cm, avg. 4.38 cm, and min 3.24 cm. The samples were contained in transparent 0.5 mL microcentrifuge tubes inserted inside 1.5 mL tubes (with some paper tissue in between them) or in transparent 200 µL PCR tubes with domed caps inserted in 500 µL tubes. For a same rotor speed, changing the sample-to-rotor axis distance results in another number of g. The sedimentation rate of the particles varies with the viscosity and density of the solvent. 2.4. Biochemical Analyses. Prior to solubility measurements, mother liquors were cleared of precipitate and crystal. The content of each sample tube was homogenized by stirring and insoluble material settled in 5 min at 1000 g (2000 rpm) in a table top centrifuge. These steps were performed at the temperature of crystallization. The supernatant was diluted twice with water and the absorbance measured on 2 µL aliquots in a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Supersaturation σ is expressed as C/S, i.e., the quotient of particle concentration C on solubility S. 2.5. X-ray Diffraction Analysis. Crystals mounted in Lindemann glass capillaries were analyzed on a Nonius diffractometer equipped with a Rigaku rotating copper anode (operated at 45 kV and 90 mA). Sets of diffraction images (oscillation angle 1°, exposure 5-20 min, crystal-to-detector distance 120 mm, diffraction limit at the edge 2.4 Å) were collected on a DIP2000b MacScience image plate. Reflections were indexed and scaled using the HKL suite of programs (DENZO/ SCALEPACK).15
3. Results 3.1. Crystallization under Hypergravity. Initial attempts to grow cubic TBSV crystals with a dodecahedral habit in the centrifuge were based on crystallization conditions taken from phase diagrams established with ammonium sulfate or PEG8000.14 Rotor speed and duration of centrifugation were derived from the sedimentation coefficient of the virions (Table 1). A first series of virus samples at 15 mg mL-1 contained in ultracentrifuge tubes maintained for 4 days at 4000 g and 4 °C produced a disk of precipitated material in solutions containing ammonium sulfate. In subsequent experiments at reduced rotor speed, nonbirefringent dodecahedral crystals measuring 300 µm on the edge appeared in the presence of above salt or PEG8000 (images a and c in Figure 1). These conditions were then transposed to a rotor that accommodates inexpensive microcentrifuge tubes and provides accelerations in the 1000-1500 g range (Table 2). Closer inspection revealed that virus crystals were truncated. The top (110) facet was inordinately large and bottom side in contact with the dense fluid was always flat. The crystals were not attached to the surface of the fluid but glided on it when the tubes were tilted (Figure 1c). On the basis of these results, conditions were searched that produce thaumatin and lysozyme crystals in the centrifuge but
not in corresponding control experiments. A few trials were sufficient to prepare birefringent crystals at previously identified precipitant and protein concentrations. In each case, for a given precipitant concentration, the above criterium was met within a narrow protein concentration range (Table 2). Optimal conditions reproducibly yielded as little as one or only a few voluminous crystals at gravity levels between 15000 and 22000 g (Figure 1). 3.2. Crystals Grown under Hypergravity. Crystals prepared in the centrifuge were stable under normal gravity. Unlike the isotropic virus crystals, those made of protein exhibited a strong birefringence in polarized light. They did not dissolve nor continued to grow as long as the temperature was the same as that of the crystallization. Most crystals prepared under hypergravity had sharp edges. Unlike the highly symmetrical virus dodecahedra, the protein crystals were not randomly oriented. The c axis of tetragonal hen lysozyme crystals was parallel to the gravity field. The pyramidal face with its four (101) facets was visible on top of the crystals (images b and d in Figure 1). The c axis of tetragonal thaumatin dipyramids that joints the apexes was perpendicular to the gravity field and the edges were not rectilinear but curved (Figure 1f). Turkey lysozyme crystals were partially embedded in the cushion of silicone fluid. Their shape diverged from that of hexagonal prisms. Neither (11j 02) nor (11j 00) facets were recognizable (Figure 1e). For the four model particles, excessive gravity always ended in crystals that were very flat and cracked (Figure 1b). Virus and protein solubilities measured at the end of the centrifugation significantly differed from those of controls (Table 3). X-ray diffraction analyses confirmed that the unit cells of the protein crystals prepared in the centrifuge are identical to those of reference crystals (Table 4). On a rotating anode the diffraction limit of the former was at least as high as that of the corresponding control. For the biggest ill-shaped turkey lysozyme crystals displayed in Figure 1e, it was 2.5 Å. The apparent mosaicity of the latter was 0.27° as compared to 0.74° for the control. 4. Discussion The high purity of the virus and protein batches chosen for the present crystallizations in an ultracentrifuge is the first difference with those reported in literature. The second difference is the use of a cushion of a colorless and chemically neutral, dense and immiscible liquid (either a silicone or a fluorocarbon) to prevent crystals from attaching to the bottom wall of the centrifugation tube, alike in container-less protein crystallization assays.16 Another one is the smaller sample volume (100 µL) as compared to the 14 and 38 mL in the case of tetrameric mammalian and fungal catalases, respectively.8,9 With inexpensive transparent PCR tubes, crystallization is feasible on as little as 10 µL solution deposited on the same volume of silicone or fluorocarbon fluid. This is close to the 4 µL horse spleen
2966 Crystal Growth & Design, Vol. 8, No. 8, 2008
Lorber
at 4 °C, solutions containing 8-10 mg mL-1 virus and 5-6.2 mM PEG-8000 having a composition very near to that on the solubility line and do not crystallize.14 Under unit gravity nucleation occurs neither at saturation (σ ) 1) nor when 1.1 < σ < 1.5. Supersaturations as low as 1.6 e σ e 3.1 provide a sufficient driving force for nucleation under hypergravity (Table 3). This means that the initial composition of the protein/precipitant mixtures is metastable, i.e., above solubility but below supersolubility (Figure 2). The latter is defined as the minimal supersaturation at which nucleation occurs spontaneously for a given solvent composition in a given environment. In the metastable zone, nuclei grow as long as the solubility is not reached. During the centrifugation of a macromolecular solution, the concentration of small solutes (such as buffer or precipitant) remains constant but larger particles move away from rotor axis and concentrate in a small volume. As a consequence, the solution gradually depletes in its upper part and enriches in particles in its lower part. A particle concentration gradient establishes that moves away from rotor axis. At a given precipitant concentration, supersaturation can exceed that at supersolubility when particle concentration is sufficient (Figure 2). When a crystal grows, the concentration of soluble particles decreases until it equals solubility. At excessive supersaturation, the elevated nucleation rate leads either to a great number of small crystals or particles become insoluble and precipitate (Figure 1). Assuming that an amount of soluble particles equal to the solubility S remains in equilibrium with the sedimented material, the maximal particle concentration at the bottom of the centrifuge tube can be approximated by
Cmax ) (Cinit - S) × 1/φ Figure 1. Virus and protein crystals grown in hypergravity. (a) Truncated dodecahedral TBSV crystals after 91 h in a 0.35 M ammonium sulfate solution at 15 mg mL-1 under an average gravity of 1000 g (AT3 rotor, 230 µL polycarbonate tube, 5000 rpm, 4 °C). A precipitate is visible as a dark layer above the silicone fluid. (b) Flat and partially cracked hen lysozyme crystals after 71 h at 1500 g in a 14 mg mL-1 solution containing 0.8 M NaCl (S45A rotor, 200 µL PCR tube, 18000 rpm, 20 °C). (c) Close-up view of a large flattened TBSV crystal prepared as in (a). Plumes of insoluble virus ascend during tube handling. (d) Single birefringent tetragonal hen lysozyme crystal on the cushion of silicone fluid produced under the same experimental conditions as in (b) but at an initial protein concentration of 12 mg mL-1. (e) Turkey lysozyme crystals embedded in silicone fluid and surrounded by microcrystalline or amorphous material after 89 h at 22000 g in a 8 mg mL-1 containing 1.7 M NaCl (S45A rotor, 500 µL microcentrifuge tube, 20000 rpm, 20 °C). The crystals have sunk into the cushion under their own weight because their density is slightly above that of the latter. The edge of the tube is seen as a dark line on the left-hand side. (f) Deformed but birefringent thaumatin dipyramides after 71 h at 15000 g in a solution containing 18 mg mL-1 protein and 0.6 M sodium tartrate (S45A rotor, PCR tube, 18000 rpm, 20 °C). The side in contact with the silicone fluid is flat. The arrow indicates the direction of gravity field G. Centrifuge tubes have been tilted backward by an angle of 45-60° to view the content. Images are slightly blurred due to the thickness and curvature of the plastic material. Scale bar is 1 mm.
apoferritin and ferritin solution used in thin glass tubes.17,18 Finally, this is the first report of the use of the viscous polymer PEG as a precipitant in hypergravity. 4.1. Crystal Nucleation under Hypergravity. The particle concentrations used here to nucleate at low precipitant concentration under hypergravity seem close to those used in conventional assays where equilibration is achieved by vapor diffusion, dialysis or counter-diffusion. Actually, they were insufficient to produce crystals in controls at 1 g. Indeed, in the phase diagram of TBSV
(1)
where Cinit is the initial concentration and φ the macromolecular volume fraction.11 For the model particles, Cmax ranges from 500 to 950 mg mL-1 or 1- to 2-fold the concentration in a crystal containing 50% solvent (Table 3). The derived maximal supersaturation σmax is 30- to 100-fold greater than that in the bulk of the starting solution and explains why nucleation is faster and more frequent in centrifuged assays. A striking feature of crystallization in the centrifuge is that the accelerations at which crystals grow are insufficient to sediment small protein monomers. A first piece of evidence is that theoretically the sedimentation of thaumatin, turkey lysozyme and hen lysozyme monomers and dimers would take about 100 h, i.e., the entire duration of the centrifugation experiments. Because crystals grow in this time span, the particles reach the bottom of the solution much faster. The sedimentation velocity of the latter can be estimated from the time they need to travel across the solution. If the time would be only one tenth of the duration of the run, then the smallest particles would have the size of aggregates composed of about 30 thaumatin or 50 lysozyme molecules. This may seem to be in contradiction with data from dynamic light scattering, small-angle X-ray scattering and analytical ultracentrifugation studies according to which hen lysozyme is essentially monomeric and thaumatin in great part dimeric in slightly supersaturated solutions.19–22 But above analyzes have also detected minute amounts of protein oligomers and greater aggregates at elevated supersaturations similar to that existing at the bottom of centrifuged solutions. The present results suggest that these prenucleation clusters accelerate mass transport in the direction of the gravity field. In this manner, they contribute to raising the particle concentration more quickly in the direction of the force field while diminishing that in the opposite direction. The gradually increasing particle concentration probably favors cluster formation as long as no nucleation event has occurred.
Virus and Protein Crystallization under Hypergravity
Crystal Growth & Design, Vol. 8, No. 8, 2008 2967
Table 2. Crystallization Conditions of Model Particles under Hypergravitya particle and sample tube
T (°C)
C (mg mL-1)
rotor type, duration, gravity levelb (speed)
precipitant, buffer, pH, crystal habit, and dimensionsc
TBSV 230 µL tube
4
15
500 µL tube
4
8
4
10
S100-AT3, 91 h 3000 g (9000 rpm) 1000 g (5000 rpm) S45A, 71 h 1500 g (6000 rpm) S45A, 91 h 1000 g (5000 rpm) S45A, 71 h 1500 g (6000 rpm) S45A, 114 h 1000 g (5000 rpm)
0.35 M ammonium sulfate, 0.05 M sodium acetate, pH 4.5 flat nonbirefringent dodecahedra, 0.3-0.4 mm across flat dodecahedra, 0.6-0.8 mm across 5 mM PEG-8000, 0.05 M sodium acetate, pH 4.5 flat dodecahedra, 0.25 × 0.25 mm2 across 5 mM PEG-8000, 0.05 M sodium acetate, pH 4.5 flat dodecahedra, ∼0.2 × 0.2 mm2 across 5 mM PEG-8000, 0.05 M sodium acetate, pH 4.5 flat dodecahedra, ∼0.3 mm across 6.2 mM PEG-8000, 0.05 M sodium acetate, pH 4.5 less flat dodecahedra, ∼0.3 mm across
S45A, 17000 S45A, 15000
114 h g (18000 rpm) 71 h g (18000 rpm)
0.6 M sodium tartrate, 0.1 M ADA, pH 6.5 deformed dipyramides, 0.15-0.2 mm long 0.6 M sodium tartrate, 0.1 M ADA, pH 6.5 deformed dipyramides 0.2-1 mm long
S45A, 89 h 22000 g (20000 rpm) 22000 g (20000 rpm)
1.7 M NaCl, 0.1 M sodium acetate, pH 4.5 deformed prisms, up to 1 mm across microcrystals
S45A, 22000 S45A, 17000 S45A, 15000
0.57 M NaCl, 0.1 M sodium acetate, pH 4.5 tetragonal crystals, ∼0.2 mm across 0.8 M NaCl, 0.1 M sodium acetate, pH 4.5 tetragonal crystals, ∼0.2 mm across 0.8 M NaCl, 0.1 M sodium acetate pH 4.5 tetragonal crystals, up to 0.8 mm across
thaumatin 500 µL tube
20
8-10
200 µL tube
20
10-18
20
8
20
10
20
18
20
10-12
20
12-14
turkey lysozyme 500 µL tube
hen lysozyme 500 µL tube
200 µL tube
67 h g (20000 rpm) 114 h g (18000 rpm) 71 h g (18000 rpm)
a All controls kept at normal gravity (1 g) produced neither crystal nor visible precipitate. b Accelerations are rounded to closest thousand. control experiments at 1 g were soluble at the end of the corresponding experiments done in the centrifuge.
c
All
Table 3. Virus and Protein Solubility and Supersaturation in Solutions Yielding Crystals under Hypergravity vs Normal Gravitya precipitant and temperature
Cinitb (mg mL-1)
φ
gravityc (g)
Sd (mg mL-1)
σ
Cmaxe (mg mL-1)
σmaxf
σmax/σ
TBSV
5 mM PEG-8000, 4 °C
8
0.006
108
16
0.012
500
50
31
turkey lysozyme
1.7 M NaCl, 20 °C
8
0.006
700
184
88
hen lysozyme
0.8 M NaCl, 20 °C
12
0.009
778
156
65
14
0.010
2.9 1.0 1.6 1.2 2.1 1.0 2.4 1.2 3.1 1.5
310
0.6 M Tartrate, 20 °C
2.8 8.0 10.0 13.6 3.8 8.0 5.0 10.3 4.5 9.6
867
thaumatin
1000 1 18000 1 22000 1 18000 1 18000 1
950
211
68
particle
a All experiments in the centrifuge produced crystals and all controls at 1 g kept at the same temperature were soluble during the whole duration of the study. b Experimental error on particle concentration is estimated to 5%. c Error on solubility is estimated to 10%. d Calculated using eq 1 (see text). e Calculated as Cmax/S.
The compressibility of the solvent at high angular velocity ω (in s-1) generates a pressure that may affect the sedimentation behavior of the macromolecules.23,24 Pressure at a depth x (in m) is given by
P ) (Fω2/2) (x2 - x02) + P0
(2)
where F is the density of the solution (kg m-3), x0 the distance from axis to meniscus (m), and P0 the pressure at the air-liquid meniscus (usually 0.1 MPa or 1 atm).19 In above assays, the pressure did not exceed 1 MPa (10 atm). Because of the greater rotor radius and the taller liquid column, it was up to 30 MPa at the bottom of the 38 mL samples of Penicillium catalase solution.7,8 Pressure may be an interesting parameter to explore. Indeed, independent experiments performed at normal gravity revealed that thaumatin crystals prepared under a pressure of 30 MPa exhibit sharper Bragg reflection profiles than controls kept at 0.1 MPa. This was explained by the fact that pressure
might dissociate unspecific aggregates that are potential sites for heterogeneous nucleation and cause defects in crystal lattices.25,26 4.2. Accelerated Crystal Growth. Earlier investigations suggested that crystal growth may be accelerated in hypergravity. Catalase crystallization with MPD and ammonium sulfate was more reproducible and up to 10-times faster when done in the centrifuge.7 Under the experimental conditions selected in the frame of the present study, assays did not produce crystals simultaneously under hypergravity and under 1 g. Even though this makes a rigorous comparison impossible, a partial answer to the question whether growth rate may be greater in the centrifuge can be given. Under normal conditions, the crystallization mechanism of TBSV proceeds by heterogeneous nucleation in a precipitate which is followed by crystal growth via Ostwald ripening.11 Here, single crystals measuring ∼0.8 mm across could be grown
2968 Crystal Growth & Design, Vol. 8, No. 8, 2008
Lorber
Table 4. Characterization of Crystals Grown under Hypergravity vs. Normal Gravitya particle thaumatin
precipitant 0.6 M tartrate
turkey lysozyme 1.7 M NaCl hen lysozyme
0.8 M NaCl
gravity session C (mg mL-1) space group 1 g, 48 h 15000 g, 71 h 1 g, 89 h 22000 g, 89 h 1 g, 114 h 15000 g, 71 h
22 16 12 8 16 12
P41212 P6122 P43212
a a a a a a
) ) ) ) ) )
b b b b b b
) ) ) ) ) )
cell parameters
crystal densityb (mg mL-1)
) ) ) ) ) )
1.23
58.6 58.7 71.4 71.2 79.8 79.4
Å, Å, Å, Å, Å, Å,
c c c c c c
151.9 Å 151.7 Å 83.1 Å, γ ) 120° 83.0 Å, γ ) 120° 38.5 Å 38.1 Å
1.23 1.11
a All crystallizations were performed at 20°C and all centrifugations were done in the S45A rotor. Crystals having approximately the same volume were analyzed in each pair of experiments. b Crystals in control and in centrifuged assays have the same density within the limits of experimental error. Large crystals of turkey lysozyme were partially sunk in the cushion because their density is very close to that of the silicone oil (d ) 1.25 mg mL-1). The effect is minimized when a liquid with a higher density is used, as for instance a fluorocarbon with d ) 1.94 mg mL-1. Smaller thaumatin crystals with the same density as those of lysozyme are lighter. The density of TBSV crystals is ∼1.16 mg mL-1.
ation (occurring during a 3 h long centrifugation) and growth (lasting 2 days outside the centrifuge).18 4.3. Crystal Quality. Macromolecular crystals that grow during centrifugation can adopt irregular shapes. Flat TBSV dodecahedra, deformed turkey lysozyme prisms, and distorted thaumatin dipyramides add to ill-shaped catalase crystals. In spite of the alteration of the habit, the latter exhibited birefringence and diffracted X-ray to 2 Å resolution. 6–8 Here, X-ray diffraction analyses indicated that the unit cells of the crystals of three other proteins were not altered by strong gravity fields. The diffraction limit and the apparent crystal mosaicity were at least as good as those of control crystals (Table 4). 5. Conclusion and Perspectives
Figure 2. Effect of centrifugation on metastable virus and protein solutions. Arrows in the two-dimensional crystallization/solubility diagram show how centrifugation changes the concentration of particles at constant number of g, temperature, and precipitant concentration. The particle concentration is shifted from its initial value Cinit (at which the composition of the solution is in the metastable zone between solubility and supersolubility lines where crystals can grow but not nucleate) to a maximal value Cmax in the nucleation zone beyond the supersolubility line. There, nucleation occurs spontaneously at moderate supersaturation. Higher particle or precipitant concentrations may favor precipitation. The concentration of soluble particles in equilibrium with crystals decreases as the latter grow and it equals solubility when crystal growth has ceased.
in only 4 days in the centrifuge as compared to 35 days outside.11 Assuming that crystal growth was continuous, the maximal growth rate would have been nine times greater in hypergravity. This is similar to what was found with catalase, for which the volume of the crystals was a function of initial concentration but not of rotor speed.7 As to the crystals of the model proteins, no effect of hypergravity on their growth rate could be detected. Turkey lysozyme crystals that measured ∼1 mm across after 89 h at 22000 g (Cinit ) 8 mg mL-1 in 1.7 M sodium chloride and at 20 °C) had grown at a rate of ∼20 Å s-1 or 8 µm h-1. Under 1 g crystals reached the same size in the same time in otherwise identical conditions but at a protein concentration of 12 mg mL-1. Similarly, in the case of hen lysozyme a 14 mg mL-1 solution subjected to 15000 g for 71 h yielded crystals measuring 0.8 mm at 20 °C and the unit gravity control crystals grew with comparable rates at 16 mg mL-1. Finally, at 17000 g and 20 °C, up to 0.2 mm long dipyramides grew within 114 h at 8 to 10 mg mL-1 thaumatin in 0.6 M tartrate, whereas controls required a protein concentration of 12 mg mL-1. It has been reported that in the cases of apoferritin and ferritin quasiequidimensional crystals can be prepared by decoupling nucle-
Pioneer investigators noted that crystallization in a centrifuge is an elegant and noninvasive method to produce well-diffracting crystals with only little equipment. Here, we have shown that this holds for biological particles with a wide range of sizes, from large viruses to small monomeric proteins. Several potential benefits are expected from strong (>1000 g) gravity fields.27 As demonstrated above, one is the control of the onset of nucleation and of the duration of crystal growth. In some cases, the rate of crystal growth could be accelerated with respect to that under normal gravity. Even though the crystal habit may be altered, the unit cell was unchanged and the diffraction limit and apparent crystal mosaicity at least as good as that of control crystals. Investigations are now needed to verify if crystal quality in terms of diffraction limit and sharpness of reflection profiles may be enhanced when crystal nucleation and growth take place under hypergravity. On the other hand, natural impurities or exogenous macromolecules displace the solubility and supersolubility in an unforeseeable manner toward higher values.28 The very pure samples employed here were not suitable to test to what extent differential sedimentation rates and hydrodynamic properties minimize the effects of macromolecular contaminants. Octahedral crystals of apoferritin grown in cadmium sulfate solution within 5 h at 2200 g in the presence of a 1000-fold molar excess of lysozyme illustrate that this is possible.17 Biochemical and crystallographic analyses should confirm if contaminants can be excluded from crystal lattices during centrifugation. Finally, a method might be implemented to search for crystallization conditions by running in parallel great numbers of assays on mixtures of precipitant and macromolecules. By combining purification and crystallization in a single operation together with accelerated crystal nucleation and growth, hypergravity would become very attractive for dilute protein samples of low purity. Acknowledgment. The author dedicates this article to the memory of Hele`ne Metzger-Bruhl for her dissertation on “La
Virus and Protein Crystallization under Hypergravity
Gene`se de la science des cristaux” defended in 1918. He has appreciated the advice of J. Witz and G. Eriani; the kind help of G. Bec, Ph. Dumas, and C. Sauter; the continued interest of R. Giege´; and the discussions with J. Kondo (IBMC) and A. Marquette (ISIS, Strasbourg). He thanks the three reviewers for their constructive comments. Support from Universite´ Louis Pasteur and from French Ministry for Research (Grants ACIBCMS 042358 and PIR-MME) is greatly acknowledged.
References (1) Ducruix, A.; Giege´, R., Eds.; Crystallization of Nucleic acids and Proteins; IRL Press: Oxford, U.K., 1999; pp 1-435. (2) McPherson, A. Crystallization of Biological Macromolecules; Cold Spring Harbor Press: Cold Spring Harbor, NY, 1999; pp 1-586. (3) Taleb, M.; Didierjean, C.; Jelsch, C.; Mangeot, J. P.; Capelle, B.; Aubry, A. J. Cryst. Growth 1999, 200, 575–582. (4) Wakayama, N. I. Cryst. Growth Des. 2003, 3, 17–24. (5) Snell, E. H.; Helliwell, J. R. Rep. Prog. Phys. 2005, 68, 799–853. (6) Vergara, A.; Lorber, B.; Sauter, C.; Giege´, R.; Zagari, A. Biophys. Chem. 2005, 118, 102–112. (7) Karpukhina, S. Y.; Barynin, V. V.; Lobanova, G. M. SoV. Phys. Crystallogr. 1975, 20, 417–418. (English translation in Kristallographia, 1975, 20, 680-681) (8) Barynin, V. V.; Melik-Adamian, W. R. SoV. Phys. Crystallogr. 1982, 27, 588–591. (English translation in Kristallographia, 1982, 27, 981987) (9) Vainshtein, B. K.; Melik-Adamyan, W. R.; Barynin, V. V.; Vagin, A. A.; Grebenko, A. I.; Borisov, V. V.; Bartels, K. S.; Fita, I.; Rossman, M. G. J. Mol. Biol. 1986, 188, 49–61.
Crystal Growth & Design, Vol. 8, No. 8, 2008 2969 (10) Lenhoff, A. M.; Pjura, P. E.; Dilmore, J. D.; Godlewski, T.S., Jr. J. Cryst. Growth 1997, 180, 113–126. (11) Lorber, B.; Witz, J. Cryst. Growth Des. 2008, 8, 1522–1529. (12) Kadri, A.; Jenner, G.; Damak, M.; Lorber, B.; Giege´, R. J. Cryst. Growth 2003, 257, 390–402. (13) Kadri, A.; Damak, M.; Lorber, B.; Giege´, R.; Jenner, G, High Pressure Res. 2003, 23, 485–491. (14) Kadri, A.; Damak, M.; Jenner, G.; Lorber, B.; Giege´, R. J. Phys.: Condens. Matter 2003, 15, 8253–8262. (15) Otwinovski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307–336. (16) Lorber, B.; Giege´, R. J. Cryst. Growth 1996, 168, 204–215. (17) Dimitrov, I.; Nanev, C. N. Cryst. Res. Technol. 2006, 41, 1063–1066. (18) Nanev, C. D.; Dimitrov, I.; Hodjaoglu, F. Ann. N.Y. Acad. Sci. 2006, 1077, 172–183. (19) Muschol, M.; Rosenberger; F, J. Cryst. Growth 1996, 167, 738–747. (20) Ducruix, A.; Guilloteau, J. P.; Rie`s-Kautt, M.; Tardieu, A. J. Cryst. Growth 1996, 168, 28–39. (21) Yoshizaki, I.; Sakai, M.; Matsuura; Y, Acta Crystallogr., Sect. D 2005, 61, 755–758. (22) Juarez-Martinez, G.; Garza, C.; Castillo, R.; Moreno, A. J. Cryst. Growth 2001, 232, 119–131. (23) Harrington, W. F.; Kegeles, G. Methods Enzymol. 1973, 27, 306– 345. (24) Schuck, P. Biophys. Chem. 2004, 108, 201–214. (25) Sear, R. P. J. Phys. Condens. Matter 2007, 19, 1–28. (26) Charron, C.; Robert, M.-C.; Capelle, B.; Kadri, A.; Jenner, G.; Giege´, R.; Lorber, B. J. Cryst. Growth 2002, 245, 321–333. (27) Schlichta, P.J. J. Cryst. Growth 1992, 119, 1–7. (28) Skouri, M.; Lorber, B.; Giege´, R.; Munch, J. P.; Candau, S. J. J. Cryst. Growth 1995, 152, 209–220.
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