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Control of nucleation and growth in protein crystal growth
74
Journal of Crystal Growth 90(1988) 74—78 North-Holland, Amsterdam
CONTROL OF NUCLEATION AND GROWTh IN PROTEIN CRYSTAL GROWTH Franz ROSENBERGER and Edward J. MEEHAN Center for Microgravity and Materials Research, and Department of Chemistry. University ofAlabama in Huntsville, Huntsville, Alabama 35899, USA
Received 25 July 1987; manuscript received in final form 7 November 1987
The potential advantages of nucleation and growth control through temperature, rather than the addition of precipitants or removal of solvent, are discussed. A simple light scattering arrangement for the characterization of nucleation and growth conditions in solutions is described. The temperature dependence of the solubility of low ionic strength lysozyme solutions is applied in preliminary nucleation and growth experiments.
1. Introduction The inability to routinely grow crystals of sufficient size and quality may be considered the major bottleneck in the further widespread development of the field of protein crystallography [1]. As pointed out by numerous workers and recently summarized in comparison with inorganic crystal growth by one of the authors [2], the fundamental understanding of the dynamics and kinetics of protein crystallization is still rather rudimentary. Correspondingly, the existing protein crystal growth technology provides only for very limited control of essential growth parameters such as number and timing of nucleation events, and growth rate. Most protein crystal growth experiments to-date result in a large number of small crystals, often too small for efficient X-ray structure studies. Though there may be some differences in complexity of the specific processes, it is probably safe to say that protein crystallization is governed by the same physico-chemical principles as inorganic crystal growth. Hence, the application of these principles and the resulting techniques to protein crystal growth can be expected to foster significant progress in this area, This paper presents a discussion of the potential of temperature as an important control
parameter in protein crystal growth. In the following sections we will: (a) summarize why temperature control has been underutilized in the past; (b) discuss the potential advantages of nucleation and growth control through temperature over current practice; (c) report preliminary results on temperature controlled protein nucleation, growth and dissolution phenomena. It is hoped that this communication will entice more protein crystal growers to consider the control of growth parameters through their temperature dependence as an advantageous alternative to the traditional approach.
2. Supersaturation control and current practice Since the pioneering work of Ostwald in 1897 [3] it is well known that the formation of a solid nucleus within a solution or melt requires a considerably higher supersaturation or undercooling, respectively, than the growth onto a solid in contact with the same nutrient. This fundamental fact forms the key to the controlled growth of large inorganic single crystals that form the mainstay of modern solid-state technology. These large in-
0022-0248/88/$03.50 © Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
F. Rosenberger, E.J. Meehan
/ Control ofnucleation
organic crystals are obtained either by: (a) supersaturating a nutrient to an extent that results in nucleation and then reducing the supersaturation such that only growth of these initial nuclei continues; or more typically by (b) contacting (“ seeding”) a saturated solution with a solid of similar or the same structure/composition and raising the supersaturation until growth occurs, respectively, via hetero- or homoepitaxy. If, on the other hand, throughout the crystallization process the supersaturation is maintained at or above the critical nucleation level, a large number of crystals of various sizes will result. Such concurrent growth and nucleation can lead to: (a) perturbations in the nutrient concentration field of and consequent inhomogeneities in the “main” crystals (i.e. that nucleated earlier), (b) misoriented growth if new crystallites become attached to main crystals, and (c) formation of structural defects due to collision with other crystals. In protein crystal growth, the supersaturation required to drive nucleation and growth is traditionally established by the addition of solubilitylowering components (precipitants) and/or the removal of solvent [4,5]. This approach makes the control of supersaturation and, in particular, its reduction after nucleation, practically difficult. Hence, concurrent growth and nucleation, and consequent crowding of crystals occurs frequently, as is well evidenced by the photographs of protein crystallization results. It has long been recognized that the degree of supersaturation required to drive nucleation within solutions increases with the structural complexity of the crystallizing solid; see e.g. ref. [6]. Thus, it is not surprising to find that protein solutions when (relatively) free of particulate matter possess very high supersaturation barriers for nucleation. In lysozyme solutions, for instance, this barrier amounts, according to our experience, to several hundred percent. At high supersaturation, however, after nucleation has set in, growth occurs initially very rapidly. Such conditions are potentially very troublesome with respect to achieving high quality crystals, since the growth of crystals of high compositional uniformity requires steady conditions with respect to both, interface shape
and growth in protein crystal growth
75
(morphological stability, see ref. [2]) and growth rate (steady segregation, see ref. [7]). In inorganic crystal growth one finds that rapid growth is often associated with morphological instabilities and consequent defect formation. With faceted crystals, morphological instabilities can be readily recognized by the appearance of a depression(s) in the center region of a facet. The protein literature shows many examples of such “depressed” growth habits (which can, of course, also result from twinning). With respect to obtaining steady growth rates, high supersaturation and growth rate conditions can also be very detrimental. Due to the low diffusivities (of proteins) in solutions, concentration gradients develop about the growing crystals. On earth, these gradients are associated with (often time-dependent) solutal convection flows; for a summary of theoretical considerations and examples of solutal convection and its consequences in inorganic crystal growth systems, see ref. [2]. Recently, the presence of solutal convection has also been demonstrated in protein growth through modelling and comparison with vapor diffusion experiments [8] and through direct flow visualization [9]. At high growth rates and, hence, large concentration gradients, time-dependent convection will prevail about a crystal and by necessity lead to non-steady segregation at the growing interface and, hence, non-uniform composition of the solid [7]. In low-gravity experiments, these uncontrolled growth conditions can become somewhat mitigated. Due to a drastic reduction of the buoyancy-driven, solutal convection, wide diffusion-governed concentration depletion zones can develop about the initial (main) crystals (unless surface tension-driven convection is significant). This provides for slower growth as well as reduces the probability for concurrent nucleation in the vicinity of the main crystals in spite of the high bulk supersaturation. This may be one of the reasons for the increase in size and decrease in the number of protein crystals that were obtained in space [10,11]. One may even speculate that the high initial supersaturations used in protein crystallization are also at the root of some of the growth cessation —
76
F Rosenberger, E.J. Meehan
/ Control ofnucleation
observations [12,13]. In a hanging drop experiment, for instance, the additional supersaturation that can be achieved with vapor diffusion after nucleation may be small as compared to the initial supersaturation required for the onset of nucleation. Hence, the initial supersaturation and drop size would essentially determine the total volume of crystals obtained, and successive (and rather limited) loss of solvent via vapor diffusion would lead only to a relatively small increase of the crystal size. Obviously, this possibility needs to be investigated quantitatively,
3. Temperature as control parameter in protein crystal growth The programming of supersaturation in inorganic solution crystal growth is typically achieved through temperature programming [14—17,and references 53—57 in ref. [2]. The influence of temperature on protein solubility has been known since the early work of Green [18]. Nevertheless, control of supersaturation through temperature has been used only to a limited extent in protein crystal growth; for a few examples, see ref. [19]. Even the control of temperature in a typical protein crystal growth situation is far less rigorous than that known to be required for the growth of homogenous inorganic crystals. The hesitancy to use temperature as a control parameter may be a legacy of the history of protein isolation. Early workers were admonished to keep the temperature as low as possible during isolation to avoid denaturation and degradation by proteases [20]. The impression of some workers that proteins have only a weak temperature dependence of the solubility may have also contributed to the neglect of this parameter. Recent extensive phase diagram work with lysozyrne [21] and some solubility measurements with canavalin [22], have shed considerable light on the above claims. Fig. 1 presents the solubility data for lysozyme obtained at pH 4.5; data taken at other pH values show similar trends [21]. One can see that the claim of insignificant temperature dependence of the solubility is quite justified at the high salt concentrations traditionally used.
and growth in protein crystal growth
However, at lower salt concentrations than those typical of current growth practice (yet high enough to stabilize the protein), the solubility can strongly depend on temperature. We have experimental evidence that the solubility surface shown rises even more rapidly for salt concentrations below 1.5% (i.e. 1.5 g/100 ml). This opens new possibilities for the design of better controlled protein crystal growth experiments. Since diffusive heat transfer in aqueous solutions proceeds about a hundred times more rapidly than (salt) concentration diffusion, supersaturation can be more readily controlled (increased and decreased!) through temperature control. This is of particular importance for growth experiments in space, where concentration adjustment rates are likely governed by the slow concentration diffusion. The additional benefits that may arise from lower salt concentrations for the interface (segregation) kinetics have been pointed out earlier [2]. Another factor that is in favor of temperature rather than concentration control is the ready availability of temperature control and programming technology. In this context one should also mention elegant work to control supersaturation through the moisture content of gas flow about protein growth drops [23]. This approach can in principle also be used to program supersaturation increases and decreases. Yet, besides the intrinsically slower transfer within the solution, the auxiliary equipment to achieve this is more cornplex than for temperature control.
4. Experiments In order to demonstrate the feasability of ternperature control of protein crystal growth we have performed a series of preliminary experiments. A lysozyme solution of low salt concentration was saturated at 20°C.Thus, according to fig. 1, both an increase and a decrease in solution temperature should lead to supersaturation. The solution was contained in a small, thermostated glass cell; see fig. 2. The temperature of the cell was adjustable. For an early detection of nucleation events, we used a simple light scattering arrangement. As
F. Rosenberger, E.J. Meehan
/
Control ofnucleation and growth in protein crystal growth
pH 4.5
100
80
I40
o Il)
20
0 10 2 20 ~1~CJ
40 10
CO~
Fig. 1. Solubiity of lysozyme at pH 4.5 as a function of temperature and NaCl (precipitant) concentration. After ref. [21].
depicted in fig. 2, a low-power laser beam was expanded into a light plane by a cylinder lens, and a 25 x long working distance microscope was focused onto and oriented normal to the light
77
plane. Experiments with this system showed that: (a) The supersaturation of approximately 100% that can be obtained by temperature changes in this specific lysozyme solution, when still and relatively free of particulates, is not sufficient to stimulate nucleation. This was to be expected in view of the high supersaturation found necessary earlier. (b) Nucleation can be induced readily upon ternperature variation (increase or decrease in this system) combined with mechanical perturbations as they result, for instance, from spinning of the stirring bar (2 in fig. 2). Again, this was to be expected since the reduction of nucleation thresholds by mechanical agitation of solutions has been known for a long time [6]. (c) Initial rapid growth at higher supersaturations (yet low as compared to common practice) can lead to morphological instabilities, such as fibrous growth. (d) There appeared to be significant asymmetries in the growth versus dissolution kinetics. Times required to dissolve crystals through temperature increase or decrease, respectively, far exceed those for growth. a sensitive means of detecting small solids in the solution. As a fringe benefit, we found that considerably more efforts than expected were required to re(e) The simple light scattering setup used provides
- - - -.
3
-
2
that conventional move were particulates thought means, from towere beafter the“clear” found solutions. to as judged beSolutions full by of scattering centers even extensive filtration
conditions and centrifugation. many speculate protein This insight crystals is nucleate important, on berecause one may that under traditional sidual particles rather than through homogeneous nucleation. This may have severe consequences with respect to straining of the crystals.
~ Fig. 2. Simple light scattering setup for observation of nucleation and growth/dissolution of protein crystals on temperature change: (1) double wall, thermostated glass cell; (2) stirring bar; (3) laser beam; (4) cylinder lens for expansion of the beam to a light plane (5); (6) long focal length microscope for visual detection of scattering centers and observation of protein crystallites’ morphology,
Acknowledgements Support of this research by the National Aeronautics and Space Administration under Grants NAS8-35611, NAGW-813 and by the State of Alabama is gratefully acknowledged.
78
F. Rosenberger, E.J. Meehan
/ Control of nucleation and growth in protein crystal growth
References
[12] Z. Kam, H.B. Shore and G. Feher, J. Mol. Biol. 123, 539 (1978).
[11 C.E. Bugg, J. Crystal Growth 76 (1986) 535 [2] F. Rosenberger, J. Crystal Growth 76 (1986) 618. [3] W. Ostwald, Z. Physik. Chem. 22 (1897) 302. [4] A. McPherson, Preparation and Analysis of Protein Crystals (Wiley, New York 1982). [5] H.W. Wyckoff, C.H.W. Hirs and SN. Timasheff, Eds., Methods in Enzymology, Vol. 114 (Academic Press, Orlando, FL, 1985). [61 A. Neuhaus, Chemie-Ing. Techn. 28 (1956) 155. [7] F. Rosenberger, Fundamentals of Crystal Growth (Springer, Berlin 1979) ch. 6. [8] WA. Fowlis, Li. DeLucas, P.J. Twigg, SB. Howard, E.J. Meehan, Jr. and J.K. Baird, J. Crystal Growth 90 (1988) 117. [9] ML. Pusey, W.K. Witherow and Ri. Naumann, J. Crystal Growth 90 (1988) 105. [10] J.L. DeLucas, FL. Suddath, R. Snyder, R. Naumann, MB. Broom, M. Pusey, V. Yost, B. Herren, D. Carter, B. Nelson, E.J. Meehan, A. McPherson and CE. Bugg, J Crystal Growth 76 (1986) 681. [11] W. Littke and Chr. John, J. Crystal Growth 76(1986) 663.
[13] G. Feher, J. Crystal Growth 76, 545 (1987). [14] T.G. Petrov et al., Growing Crystals From Solution (Consultants Bureau, New York, 1969). [15] J.W. Mullin, Crystallization (Butterworth, London, 1972). [16] J.A. James and R.C. Kell, in: Crystal Growth, Ed. BR. Pamplin (Pergamon, Oxford, 1975). [17] J.C. Bnce, Crystal Growth Processes (Blackie, Glasgow/ Wiley, New York, 1986) ch. 6. [18] A.A. Green, J. Biol. Chem. 93 (1931) 495. [19] A. McPherson, In: Methods in Enzymology, Vol. 114, Eds. H.W. Wyckoff, C.H.W. Hirs and SN. Timasheff (Academic Press, Orlando, FL, 1985) p. 127. [20] E.J. Cohn, in: Proteins, Amino Acids and Peptides, Eds. E.J. John and J.T. Edsall (Hafner, New York, 1943). [21] SB. Howard, P.J. Twigg, J.K. Baird and E.J. Meehan. J. Crystal Growth 90 (1988) 94. [22] CC. Young, R.C. De Mattei, R.S. Feigelson and WA. Tiller, J. Crystal Growth 90 (1988) 79. [23] B. Suddath, paper at Protein Crystal Growth Workshop, Joe Wheeler State Park, May 1987.
Journal of Crystal Growth 90(1988) 74—78 North-Holland, Amsterdam
CONTROL OF NUCLEATION AND GROWTh IN PROTEIN CRYSTAL GROWTH Franz ROSENBERGER and Edward J. MEEHAN Center for Microgravity and Materials Research, and Department of Chemistry. University ofAlabama in Huntsville, Huntsville, Alabama 35899, USA
Received 25 July 1987; manuscript received in final form 7 November 1987
The potential advantages of nucleation and growth control through temperature, rather than the addition of precipitants or removal of solvent, are discussed. A simple light scattering arrangement for the characterization of nucleation and growth conditions in solutions is described. The temperature dependence of the solubility of low ionic strength lysozyme solutions is applied in preliminary nucleation and growth experiments.
1. Introduction The inability to routinely grow crystals of sufficient size and quality may be considered the major bottleneck in the further widespread development of the field of protein crystallography [1]. As pointed out by numerous workers and recently summarized in comparison with inorganic crystal growth by one of the authors [2], the fundamental understanding of the dynamics and kinetics of protein crystallization is still rather rudimentary. Correspondingly, the existing protein crystal growth technology provides only for very limited control of essential growth parameters such as number and timing of nucleation events, and growth rate. Most protein crystal growth experiments to-date result in a large number of small crystals, often too small for efficient X-ray structure studies. Though there may be some differences in complexity of the specific processes, it is probably safe to say that protein crystallization is governed by the same physico-chemical principles as inorganic crystal growth. Hence, the application of these principles and the resulting techniques to protein crystal growth can be expected to foster significant progress in this area, This paper presents a discussion of the potential of temperature as an important control
parameter in protein crystal growth. In the following sections we will: (a) summarize why temperature control has been underutilized in the past; (b) discuss the potential advantages of nucleation and growth control through temperature over current practice; (c) report preliminary results on temperature controlled protein nucleation, growth and dissolution phenomena. It is hoped that this communication will entice more protein crystal growers to consider the control of growth parameters through their temperature dependence as an advantageous alternative to the traditional approach.
2. Supersaturation control and current practice Since the pioneering work of Ostwald in 1897 [3] it is well known that the formation of a solid nucleus within a solution or melt requires a considerably higher supersaturation or undercooling, respectively, than the growth onto a solid in contact with the same nutrient. This fundamental fact forms the key to the controlled growth of large inorganic single crystals that form the mainstay of modern solid-state technology. These large in-
0022-0248/88/$03.50 © Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
F. Rosenberger, E.J. Meehan
/ Control ofnucleation
organic crystals are obtained either by: (a) supersaturating a nutrient to an extent that results in nucleation and then reducing the supersaturation such that only growth of these initial nuclei continues; or more typically by (b) contacting (“ seeding”) a saturated solution with a solid of similar or the same structure/composition and raising the supersaturation until growth occurs, respectively, via hetero- or homoepitaxy. If, on the other hand, throughout the crystallization process the supersaturation is maintained at or above the critical nucleation level, a large number of crystals of various sizes will result. Such concurrent growth and nucleation can lead to: (a) perturbations in the nutrient concentration field of and consequent inhomogeneities in the “main” crystals (i.e. that nucleated earlier), (b) misoriented growth if new crystallites become attached to main crystals, and (c) formation of structural defects due to collision with other crystals. In protein crystal growth, the supersaturation required to drive nucleation and growth is traditionally established by the addition of solubilitylowering components (precipitants) and/or the removal of solvent [4,5]. This approach makes the control of supersaturation and, in particular, its reduction after nucleation, practically difficult. Hence, concurrent growth and nucleation, and consequent crowding of crystals occurs frequently, as is well evidenced by the photographs of protein crystallization results. It has long been recognized that the degree of supersaturation required to drive nucleation within solutions increases with the structural complexity of the crystallizing solid; see e.g. ref. [6]. Thus, it is not surprising to find that protein solutions when (relatively) free of particulate matter possess very high supersaturation barriers for nucleation. In lysozyme solutions, for instance, this barrier amounts, according to our experience, to several hundred percent. At high supersaturation, however, after nucleation has set in, growth occurs initially very rapidly. Such conditions are potentially very troublesome with respect to achieving high quality crystals, since the growth of crystals of high compositional uniformity requires steady conditions with respect to both, interface shape
and growth in protein crystal growth
75
(morphological stability, see ref. [2]) and growth rate (steady segregation, see ref. [7]). In inorganic crystal growth one finds that rapid growth is often associated with morphological instabilities and consequent defect formation. With faceted crystals, morphological instabilities can be readily recognized by the appearance of a depression(s) in the center region of a facet. The protein literature shows many examples of such “depressed” growth habits (which can, of course, also result from twinning). With respect to obtaining steady growth rates, high supersaturation and growth rate conditions can also be very detrimental. Due to the low diffusivities (of proteins) in solutions, concentration gradients develop about the growing crystals. On earth, these gradients are associated with (often time-dependent) solutal convection flows; for a summary of theoretical considerations and examples of solutal convection and its consequences in inorganic crystal growth systems, see ref. [2]. Recently, the presence of solutal convection has also been demonstrated in protein growth through modelling and comparison with vapor diffusion experiments [8] and through direct flow visualization [9]. At high growth rates and, hence, large concentration gradients, time-dependent convection will prevail about a crystal and by necessity lead to non-steady segregation at the growing interface and, hence, non-uniform composition of the solid [7]. In low-gravity experiments, these uncontrolled growth conditions can become somewhat mitigated. Due to a drastic reduction of the buoyancy-driven, solutal convection, wide diffusion-governed concentration depletion zones can develop about the initial (main) crystals (unless surface tension-driven convection is significant). This provides for slower growth as well as reduces the probability for concurrent nucleation in the vicinity of the main crystals in spite of the high bulk supersaturation. This may be one of the reasons for the increase in size and decrease in the number of protein crystals that were obtained in space [10,11]. One may even speculate that the high initial supersaturations used in protein crystallization are also at the root of some of the growth cessation —
76
F Rosenberger, E.J. Meehan
/ Control ofnucleation
observations [12,13]. In a hanging drop experiment, for instance, the additional supersaturation that can be achieved with vapor diffusion after nucleation may be small as compared to the initial supersaturation required for the onset of nucleation. Hence, the initial supersaturation and drop size would essentially determine the total volume of crystals obtained, and successive (and rather limited) loss of solvent via vapor diffusion would lead only to a relatively small increase of the crystal size. Obviously, this possibility needs to be investigated quantitatively,
3. Temperature as control parameter in protein crystal growth The programming of supersaturation in inorganic solution crystal growth is typically achieved through temperature programming [14—17,and references 53—57 in ref. [2]. The influence of temperature on protein solubility has been known since the early work of Green [18]. Nevertheless, control of supersaturation through temperature has been used only to a limited extent in protein crystal growth; for a few examples, see ref. [19]. Even the control of temperature in a typical protein crystal growth situation is far less rigorous than that known to be required for the growth of homogenous inorganic crystals. The hesitancy to use temperature as a control parameter may be a legacy of the history of protein isolation. Early workers were admonished to keep the temperature as low as possible during isolation to avoid denaturation and degradation by proteases [20]. The impression of some workers that proteins have only a weak temperature dependence of the solubility may have also contributed to the neglect of this parameter. Recent extensive phase diagram work with lysozyrne [21] and some solubility measurements with canavalin [22], have shed considerable light on the above claims. Fig. 1 presents the solubility data for lysozyme obtained at pH 4.5; data taken at other pH values show similar trends [21]. One can see that the claim of insignificant temperature dependence of the solubility is quite justified at the high salt concentrations traditionally used.
and growth in protein crystal growth
However, at lower salt concentrations than those typical of current growth practice (yet high enough to stabilize the protein), the solubility can strongly depend on temperature. We have experimental evidence that the solubility surface shown rises even more rapidly for salt concentrations below 1.5% (i.e. 1.5 g/100 ml). This opens new possibilities for the design of better controlled protein crystal growth experiments. Since diffusive heat transfer in aqueous solutions proceeds about a hundred times more rapidly than (salt) concentration diffusion, supersaturation can be more readily controlled (increased and decreased!) through temperature control. This is of particular importance for growth experiments in space, where concentration adjustment rates are likely governed by the slow concentration diffusion. The additional benefits that may arise from lower salt concentrations for the interface (segregation) kinetics have been pointed out earlier [2]. Another factor that is in favor of temperature rather than concentration control is the ready availability of temperature control and programming technology. In this context one should also mention elegant work to control supersaturation through the moisture content of gas flow about protein growth drops [23]. This approach can in principle also be used to program supersaturation increases and decreases. Yet, besides the intrinsically slower transfer within the solution, the auxiliary equipment to achieve this is more cornplex than for temperature control.
4. Experiments In order to demonstrate the feasability of ternperature control of protein crystal growth we have performed a series of preliminary experiments. A lysozyme solution of low salt concentration was saturated at 20°C.Thus, according to fig. 1, both an increase and a decrease in solution temperature should lead to supersaturation. The solution was contained in a small, thermostated glass cell; see fig. 2. The temperature of the cell was adjustable. For an early detection of nucleation events, we used a simple light scattering arrangement. As
F. Rosenberger, E.J. Meehan
/
Control ofnucleation and growth in protein crystal growth
pH 4.5
100
80
I40
o Il)
20
0 10 2 20 ~1~CJ
40 10
CO~
Fig. 1. Solubiity of lysozyme at pH 4.5 as a function of temperature and NaCl (precipitant) concentration. After ref. [21].
depicted in fig. 2, a low-power laser beam was expanded into a light plane by a cylinder lens, and a 25 x long working distance microscope was focused onto and oriented normal to the light
77
plane. Experiments with this system showed that: (a) The supersaturation of approximately 100% that can be obtained by temperature changes in this specific lysozyme solution, when still and relatively free of particulates, is not sufficient to stimulate nucleation. This was to be expected in view of the high supersaturation found necessary earlier. (b) Nucleation can be induced readily upon ternperature variation (increase or decrease in this system) combined with mechanical perturbations as they result, for instance, from spinning of the stirring bar (2 in fig. 2). Again, this was to be expected since the reduction of nucleation thresholds by mechanical agitation of solutions has been known for a long time [6]. (c) Initial rapid growth at higher supersaturations (yet low as compared to common practice) can lead to morphological instabilities, such as fibrous growth. (d) There appeared to be significant asymmetries in the growth versus dissolution kinetics. Times required to dissolve crystals through temperature increase or decrease, respectively, far exceed those for growth. a sensitive means of detecting small solids in the solution. As a fringe benefit, we found that considerably more efforts than expected were required to re(e) The simple light scattering setup used provides
- - - -.
3
-
2
that conventional move were particulates thought means, from towere beafter the“clear” found solutions. to as judged beSolutions full by of scattering centers even extensive filtration
conditions and centrifugation. many speculate protein This insight crystals is nucleate important, on berecause one may that under traditional sidual particles rather than through homogeneous nucleation. This may have severe consequences with respect to straining of the crystals.
~ Fig. 2. Simple light scattering setup for observation of nucleation and growth/dissolution of protein crystals on temperature change: (1) double wall, thermostated glass cell; (2) stirring bar; (3) laser beam; (4) cylinder lens for expansion of the beam to a light plane (5); (6) long focal length microscope for visual detection of scattering centers and observation of protein crystallites’ morphology,
Acknowledgements Support of this research by the National Aeronautics and Space Administration under Grants NAS8-35611, NAGW-813 and by the State of Alabama is gratefully acknowledged.
78
F. Rosenberger, E.J. Meehan
/ Control of nucleation and growth in protein crystal growth
References
[12] Z. Kam, H.B. Shore and G. Feher, J. Mol. Biol. 123, 539 (1978).
[11 C.E. Bugg, J. Crystal Growth 76 (1986) 535 [2] F. Rosenberger, J. Crystal Growth 76 (1986) 618. [3] W. Ostwald, Z. Physik. Chem. 22 (1897) 302. [4] A. McPherson, Preparation and Analysis of Protein Crystals (Wiley, New York 1982). [5] H.W. Wyckoff, C.H.W. Hirs and SN. Timasheff, Eds., Methods in Enzymology, Vol. 114 (Academic Press, Orlando, FL, 1985). [61 A. Neuhaus, Chemie-Ing. Techn. 28 (1956) 155. [7] F. Rosenberger, Fundamentals of Crystal Growth (Springer, Berlin 1979) ch. 6. [8] WA. Fowlis, Li. DeLucas, P.J. Twigg, SB. Howard, E.J. Meehan, Jr. and J.K. Baird, J. Crystal Growth 90 (1988) 117. [9] ML. Pusey, W.K. Witherow and Ri. Naumann, J. Crystal Growth 90 (1988) 105. [10] J.L. DeLucas, FL. Suddath, R. Snyder, R. Naumann, MB. Broom, M. Pusey, V. Yost, B. Herren, D. Carter, B. Nelson, E.J. Meehan, A. McPherson and CE. Bugg, J Crystal Growth 76 (1986) 681. [11] W. Littke and Chr. John, J. Crystal Growth 76(1986) 663.
[13] G. Feher, J. Crystal Growth 76, 545 (1987). [14] T.G. Petrov et al., Growing Crystals From Solution (Consultants Bureau, New York, 1969). [15] J.W. Mullin, Crystallization (Butterworth, London, 1972). [16] J.A. James and R.C. Kell, in: Crystal Growth, Ed. BR. Pamplin (Pergamon, Oxford, 1975). [17] J.C. Bnce, Crystal Growth Processes (Blackie, Glasgow/ Wiley, New York, 1986) ch. 6. [18] A.A. Green, J. Biol. Chem. 93 (1931) 495. [19] A. McPherson, In: Methods in Enzymology, Vol. 114, Eds. H.W. Wyckoff, C.H.W. Hirs and SN. Timasheff (Academic Press, Orlando, FL, 1985) p. 127. [20] E.J. Cohn, in: Proteins, Amino Acids and Peptides, Eds. E.J. John and J.T. Edsall (Hafner, New York, 1943). [21] SB. Howard, P.J. Twigg, J.K. Baird and E.J. Meehan. J. Crystal Growth 90 (1988) 94. [22] CC. Young, R.C. De Mattei, R.S. Feigelson and WA. Tiller, J. Crystal Growth 90 (1988) 79. [23] B. Suddath, paper at Protein Crystal Growth Workshop, Joe Wheeler State Park, May 1987.