Use of Capillaries for Macromolecular Crystallization in a Cryogenic Dewar Ewa
Ciszak,*,†,‡
Hammons,‡
Aaron S.
and Young Soo
Hong‡
NASA/Marshall Space Flight Center, Biophysical and Space Research Laboratory, Mail Code SD46, Huntsville, Alabama 35812, and Universities Space Research Association, 4950 Corporate Drive, Suite 100, Huntsville, Alabama 35805 Received November 24, 2001;
CRYSTAL GROWTH & DESIGN 2002 VOL. 2, NO. 3 235-238
Revised Manuscript Received February 14, 2002
ABSTRACT: The enhanced gaseous nitrogen (EGN) dewar is a cryogenic dry shipper with a sealed cylinder inserted inside along with a temperature-monitoring device, and is intended for macromolecular crystallization experiments on the International Space Station. Within the dewar, each crystallization experiment is contained as a solution within a plastic capillary tube. The standard procedure for loading samples in these tubes has involved rapid freezing of the precipitant and biomolecular solution, e.g., protein, directly in liquid nitrogen; this method, however, often resulted in uncontrolled formation of air voids. These air pockets, or bubbles, can lead to irreproducible crystallization results. A novel protocol has been developed to prevent formation of bubbles, and this has been tested in the laboratory as well as aboard the International Space Station during a 42-day long mission of July/August 2001. The gain or loss of mass from solutions within the plastic capillaries revealed that mass transport occurred among separated tubes, and that this mass transport was dependent upon the hygroscopic character of the solution contained in any given tube. The surface area of the plastic capillary tube also related to the observed mass transport. Furthermore, the decreased mass of solutions of protein correlated to observed formation of protein crystals. 1. Introduction Crystallization experiments aboard the International Space Station (ISS) are focused on the use of a lowgravity environment for achieving large and wellordered crystals of various materials. This provides awareness of potential value for use of the ISS platform as a tool for advancing the knowledge of biological materials including proteins, viral particles, and nucleic acids. The three-dimensional structures of these macromolecules and assemblies hold the information needed for the discipline of structural biology to contribute to the development of new medicines, materials, and diagnostic procedures. The main advantage that microgravity offers to the process of macromolecular crystallization is the possibility of achieving quiescent interfaces between mixing solutions, where those solutions are free from gradients of hydrostatic pressure, sedimentation, buoyancy, and thermal/solutal convection, and thereby promoting highly sensitive response to the weak forces of molecular interactions.1 One simple flight hardware used for macromolecular crystallization aboard the ISS is the enhanced gaseous nitrogen (EGN) dewar.2 The EGN dewar is a cryogenic dry shipper with a sealed aluminum cylinder inserted inside as pictured in Koszelak et al.2 The dry shipper is charged initially with liquid nitrogen, and therefore keeps the samples of solutions inside plastic capillaries frozen at -196 °C during transfer to the ISS and thereafter, until the nitrogen evaporates. The EGN dewar reaches ambient temperature of the ISS cabin approximately 11 days following launch. At this time, crystallization samples are contained within plastic (Tygon) capillaries, typically with a * Correspondence address: E-mail:
[email protected]. † NASA/Marshall Space Flight Center. ‡ Universities Space Research Association.
maximum volume of approximately 140 µL that by current protocol are snap-frozen directly in liquid nitrogen. Hundreds of these tubes can be accommodated in the sealed insert of the EGN dewar, and these typically employ free-interface liquid-liquid diffusion crystallization technique. Crystals produced within the EGN dewar have been used for studies of crystal morphology, internal crystal quality, or the determination of the three-dimensional crystal structure using X-ray diffraction methods.3-6 However, the current protocol for preparing and loading samples often has led to air voids or bubbles trapped in the tubes. These discontinuities can prevent completion of the process of free-interface liquid-liquid exchange, and may alter the outcome of crystallization process. Neither the location of these air gaps, nor their sizes have been controllable thus far, making experimental outcome unpredictable and impairing reproducibility needed for statistical confidence. We therefore devised a novel and simple sample preparation procedure that eliminates uncontrolled formation of bubbles within the sample solution. Experiments conducted in our laboratory also showed that the currently used Tygon capillaries lose sample mass. This loss of mass through water vapor diffusion becomes important in controlling the conditions of crystallization, equilibration rates, and the methods of storing the tubes. We therefore characterized conditions contributing to this mass transport, and established conditions to minimize that transport. Formation of uncontrolled air gaps and change of mass observed in the ground experiments indicated a need to further refine knowledge within microgravity of these observed physicochemical behaviors of crystallization samples. We therefore also extended our investigations that led to elimination of air bubbles in Tygon capillaries for further understanding of technical capabilities of macromolecular crystallization within the
10.1021/cg015567l CCC: $22.00 © 2002 American Chemical Society Published on Web 03/27/2002
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EGN dewar during a recent experiment aboard the ISS. The orbiter was launched with the EGN dewar on the 12th of July 2001, and the payload was returned on the 22nd of August 2001 after a 42-day mission. 2. Experimental Design 2.1. Samples. Four sets of samples were prepared. Each set included the following solutions: (1) 50% PEG4000 in 50 mM sodium/potassium phosphate buffer, pH ) 8.0, (2) 2 M ammonium sulfate in 50 mM sodium/potassium phosphate buffer, pH ) 8.0, (3) 40 mg/mL lysozyme prepared in 50 mM sodium acetate, pH ) 4.7, and (4) 40 mg/mL lysozyme in 50 mM sodium acetate separately added with a second precipitant solution containing 1 M sodium chloride in 50 mM sodium acetate, pH ) 4.7, prepared by the free-interface liquid-liquid diffusion method. For visual clarity of outcome, all protein solutions in series 4 only were colored with a stock solution of methylene blue dye (final addition 5% v/v) (Izit, Hampton Research). 2.2. Sample Loading Procedure. All solutions were degassed for 5 min under partial vacuum prior to loading in plastic capillaries. The tubes used were Tygon S-50-HL formulation, and two sets were prepared with different internal diameters: one set with 1.6 mm and another set with 2.4 mm diameter. The thickness of walls in both sets of tubes was 0.8 mm. The tubes were cut to a length of 10 cm. Following this step, tubes were marked with a pen 1.0 cm from each end to indicate sealing positions, and in the center of the tube to allow for accurate placement of solutions. The resulting sealed length of the capillaries was 8 cm; the maximum volume introduced to both the small and large diameter capillaries was 140 µL. When loaded for free interface liquid-liquid diffusion, precipitant solution was always loaded first, its loading end sealed with a SEBRA model 1090 sealer (Tucson, AZ), and the tube was then placed in a freezer at -80 °C. Samples froze within a few minutes. Alternatively, the tubes were at times frozen by placing on top of a bed of dry ice at -79 °C. Once the precipitant froze completely, the tube was transported on a bed of dry ice to the lab bench for addition then of the protein solution. While the sample was held over dry ice, protein solution was quickly added through the remaining open end, which was then sealed at the second sealing mark. Immediately after this process, the tube was placed into a laboratory cryogenic dry shipper at -196 °C, and stored at that temperature until transfer to the EGN dewar. When loaded for a batch method, the loading process of the premixed protein plus precipitant solutions involved sealing the second end as well at the sealing mark before freezing the tube, first at -80 °C followed then by transfer into the cryogenic dry shipper at -196 °C. It was important never to introduce the tubes directly into liquid nitrogen to avoid appearance of air bubbles. 2.3. Measurements of Mass of Samples Loaded into Tubes. Three series of tubes containing homogeneous solutions, i.e., PEG4000 (set 1), ammonium sulfate (set 2), and lysozyme (set 3), were used for measurements of the rates of change of mass following from evaporation of liquid. Series 4 contained two separate solutions in contact with each other within the tube, one being protein solution and the second being the precipitant solution, were not used in the mass measurements because they were kept frozen at all times. Frozen tubes were noted to be intractable for weighing due to condensation from the air. The volumes of solutions prepared in each series were 20, 60, 100, and 140 µL, and each of the volumes were prepared in triplicate. Each sample tube was sealed on both ends, weighed before freezing using an analytical balance from Mettler Toledo model AG204 (( 0.1 mg repeatibility), and transferred to the dewar. Within 3 h after return from the ISS, tubes were packed individually in 15mL polypropylene conical centrifuge tubes (Falcon Max, VWR), sealed, and then transported to and weighed in our laboratory within 12 h to estimate the change in mass of tubes that had occurred during the mission. Tubes were then held in a 22 °C
Figure 1. A plastic (Tygon) capillary representative of 130 tubes recovered from the EGN dewar after a 42-day space flight demonstrates results of the loading protocol that employs (1) a freeze of a precipitant at -80 °C followed by (2) addition of protein solution, and (3) a freeze at -196 °C in a cryogenic dry shipper. The protein solution was stained with methylene blue dye, and the continuity of blue color indicates completion of the diffusion process and lack of any uncontrolled air voids along the length of the solution. The total loaded volume of the solution was 100 µL. incubator, and the weighing procedure was repeated again four times after 48, 96, 192, and 240 h following the first postflight weight measurement. Weights of tubes were tracked within their individual sets, each of which were held in a different environment. Those environments included sealed 15-mL polypropylene conical tube (T), sealed plastic box (B) (Hampton Research), and unprotected on the shelf (S) of the laboratory incubator.
3. Results 3.1. Novel Loading Procedure. The new loading procedure involves first the freeze of a precipitant in the capillary at -80 °C, followed by addition of a protein and rapid freeze of the tube at -196 °C by insertion within a laboratory cryogenic dry shipper, avoiding direct exposure to liquid nitrogen at all times. Once samples accumulate in the cryogenic dry shipper, they can then be loaded into the EGN dewar for subsequent space flight. This new method compares with the previous method that called for freezing the protein first by immersing the tube in liquid nitrogen, followed by removal from liquid nitrogen, and addition of a precipitant, sealing the second end of the tube, and then immersing the tube in liquid nitrogen again.2 That previous method often led to uncontrolled formation of bubbles. In contrast, our new procedure shows that replacing liquid nitrogen with an atmosphere of cold nitrogen gas inside a cryogenic dry shipper avoids bubbles in the tubes. An example of a single thawed tube from set 4 containing a total of 100 µL of solution prepared by our new procedure is shown in Figure 1. The solution in Figure 1 was formed by diffusive mixing of initially separate precipitant and protein-methylene blue solutions. The different volumes of liquids employed, and the two diameters of the tubes tested, provided the same absence of bubbles as an outcome of this loading procedure. Out of 130 samples containing one loaded solution or two loaded solutions, prepared using this above new protocol, a single air bubble was found in only two tubes, and each of those bubbles was thought to be introduced by operator error. 3.2. Gain and Loss of Mass of Samples During Flight. The sample mass changes, dm (dm ) mpostflight - mpreflight) are shown in Figure 2 as a function of the sample volumes and the diameter of the tubes. The tubes containing PEG4000 (set 1) and ammonium sulfate (set 2) gained mass, while the tubes containing
Capillaries for Macromolecular Crystallization
Figure 2. Change in mass of solutions of 50% PEG4000 (set 1), 2 M ammonium sulfate (set 2), and 40 mg/mL lysozyme in 50 mM sodium acetate buffer, pH ) 4.7 (set 3) contained in plastic capillaries within the EGN dewar during a flight experiment conducted aboard the International Space Station. The change of mass of solutions in 1.6-mm internal diameter capillaries are shown as solid lines, while the change of mass of solutions contained in 2.4-mm internal diameter capillaries are shown as dashed lines. The PEG4000 and ammonium sulfate solutions gained mass, while the lysozyme protein solution lost mass. The change in mass, and standard deviations of the mean are shown relative to the initial volumes of solutions within each set, i.e., 20, 60, 100, and 140 µL, of the solutions.
lysozyme (set 3) solution lost mass. The increase of mass of PEG4000 solution (set 1) in 1.6-mm diameter tubes ranged from 2.1 (( 0.6) mg for 20 µL sample volume to 3.1 (( 0.6) mg for 140 µL sample volume, where errors are given as standard deviation of the mean. The increase of mass in 2.4 mm diameter tubes was larger, ranging from 3.0 (( 0.6) mg for 20 µL sample volumes to 4.5 (( 0.6) mg for 140 µL sample volumes. The gain in mass for ammonium sulfate solutions (set 2) was greater than that recorded for respective PEG4000 solutions. The gain in mass of ammonium sulfate solution ranged from 2.6 (( 0.6) mg for 20 µL sample volume to 3.6 (( 0.6) mg for 140 µL of sample volumes contained in 1.6 mm diameter tubes. The gain of mass in 2.4-mm internal diameter tubes was larger, ranging from 4.0 (( 0.7) mg for 20 µL of sample volumes to 5.1 (( 0.7) mg for 60 µL of sample volumes. In contrast, tubes containing lysozyme (set 3) in nonhygroscopic solution lost sample mass. The loss in mass ranged from 1.9 (( 0.6) mg for 140 µL sample volumes to 2.6 (( 0.7) for 60 µL sample volumes in 1.6mm diameter tubes. The loss in mass of solutions contained in 2.4-mm diameter tubes was greater, ranging from 2.4 (( 0.6) mg for 140 µL sample volumes to 3.8 (( 0.6) mg for 60 µL sample volumes. Loss of sample by evaporation through the plastic tube walls is a form of vapor diffusion technique. Not surprisingly, large lysozyme crystals were observed to develop in all tubes during the drying process (results not shown). 3.3. Loss of Mass of Samples after Flight. Once the tubes were removed from the EGN dewar insert, a process of evaporation began for all solutions that became notable after 48 h. This is shown in Figure 3. The rate of mass-loss over a 10-day period is influenced by the type of environment that the tubes were kept in. For example, the 1.6-mm diameter capillaries containing 140 µL of solution exposed to air in the incubator lost mass at the rate of approximately 1.9 mg/day, while
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Figure 3. The rate of loss of sample mass of PEG4000, ammonium sulfate, and lysozyme contained in 1.6-mm internal diameter capillary tube. Each set of three tubes from the 140 µL sample volumes of Figure 2 weighed within 12 h after return from ISS was divided so that one tube was held on the shelf (marked with O), one in a conical tube (marked with T), and one tube in a plastic box (marked with B), all in a 22 °C laboratory incubator. The temporal pattern of the loss of mass from all other sample sets was similar, including the samples from 2.4-mm diameter capillary tubes.
those placed either in sealed plastic boxes or in 15-mL conical tubes lost mass at the average rate of approximately 1.0 mg/day. The rate of loss of mass was independent of the starting volume of the sample in the tube. However, the loss of mass in pairs of tubes containing the same sample volumes of the same solutions is greater in 2.4-mm diameter tubes than in 1.6-mm tubes. For example, the average loss of sample mass after 48 h in 2.4-mm capillaries held on the shelf of the incubator was approximately 2.1 mg/day. If mass transport among plastic tubes is to be of value as a new method of crystallization, then it is important to be able to control this process completely. Interestingly, it was established that loss of sample mass was prevented when tubes were kept under a layer of mineral oil. 4. Discussion Results of mass transport support an assumption that the surface area and composition of solution contained in Tygon capillary tubes influenced the mass transport of sample volumes observed among all tubes. The direction of mass transport within the EGN dewar was determined by the hygroscopic character of the solution contained in any given tube, since the insert inside the cryogenic EGN dewar was hermetically sealed at the time of sample loading. This gain and loss of sample mass likely was accomplished through migration of water vapor, presumably driven by equilibria processes within the closed system of the EGN dewar initially, and by evaporation processes thereafter while held in the laboratory incubator. It was not possible to study offsetting gain and loss of sample mass in these tubes to confirm this assumption of thermodynamic equilibrium because the dewar insert also contained tubes flown by other investigators. Those other capillary tubes also contained numerous precipitant and protein solutions that contributed to the assumed equilibrium of mass transport in unknown ways. These observations raise a useful point, and that is that individual experi-
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ments, i.e., Tygon capillary tubes, will participate in mass transport within the insert of the EGN dewar unless they are individually isolated from that participation. Our laboratory findings suggest that a sheathe of mineral oil might be used as a simple method to provide that individual isolation. The measurements of sample mass-change within the EGN dewar shows that mass transport in the fixed geometry of tubes reached 25% for 20 µL sample volumes and significantly less for larger sample volumes, in some cases only 3.5% for 140 µL sample volumes. This observation provides additional evidence that the surface area of the tube and the volume of headspace inside the tubes around the meniscus of the sample relate to the observed mass transport. The greatest loss of mass from samples of lesser volume, e.g., 60 µL > 140 µL, as shown in Figure 2 was unexpected. This suggests that the hygroscopic character of the solutions contained in adjacent capillary tubes versus remotely located tubes within the EGN dewar may disproportionately influence the kinetics of mass transport. The EGN dewar used to support crystallization aboard the ISS has both advantages and limitations. At this point of development, the EGN dewar is best considered for robust crystallization experiments employing large volumes of sample solutions, i.e., approximately 100 µL or more, providing then large crystals and/or large quantities of crystals. Despite limitations of controls characterized here, our new loading procedure that prevents formation of air bubbles, and the new observation of vapor diffusion among tubes in the sealed insert of the EGN dewar, identify refinements to be made that can lead to use of the EGN dewar as an excellent method for growing crystals of a wide range of biomaterials aboard the ISS.
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Furthermore, we suggest that the observed vapor diffusion among Tygon tubes, apparently driven by approach to thermodynamic equilibrium, introduces a new potentially useful method of crystallization. It has been common practice thus far to introduce standard laboratory procedures of crystallization into microgravity studies. However, this evaluation of plastic capillaries from a microgravity study appears to possibly introduce new technique for vapor diffusion crystallization method into standard laboratory practice. That possibility is supported by observations of appearance of numerous, rather large lysozyme crystals appearing in all tubes with protein solutions during postflight storage. Acknowledgment. This work was supported in part by the grants from the National Space Science and Technology Center (NSSTC-004), National Aeronautics and Space Administration (NAG-1841), and by the EGN Dewar project funds made available through Ray French, the EGN Project Manager. The authors wish to thank Dr. Robert Richmond for helpful discussions. References (1) Martinez, I.; Haynes, J. M.; Langbein, D. In Fluid Sciences and Materials Science in Space; Walter, H. U., Ed.; 1987; pp 53-81. (2) Koszelak, S.; Leja, C.; McPherson, A. Biotechnol. Bioeng. 1996, 52, 449-458. (3) Alvarado, U. R.; DeWitt, C. R.; Shulz, B. B.; Ramsland, P. A.; Edmundson, A. B. J. Cryst. Growth 2001, 223, 407-414. (4) McPherson, A. Tibtech. 1997, 15, 187-200. (5) Ko. T.-P.; Day, J.; Malkin, A. J.; McPherson, A. Acta Crystallogr. 1999, D55, 1383-1394. (6) Ko, T.-P.; Kuznetsov, Y. G.; Malkin, A. J.; Day, J.; McPherson, A. Acta Crystallogr. 2001, D57, 829-839.
CG015567L
