Michelson interferometric studies of protein and virus crystal growth

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ELSEVIER

CRYSTAL GROWTH

Journal of Crystal Growth 166 (1996) 913-918

Michelson interferometric studies of protein and virus crystal growth Yu. G. Kuznetsov *, A.J. Malkin, A. Greenwood, A. McPherson Department of Biochemistry, University of California, Riverside, California 92521, USA

Abstract

In situ laser Michelson interferometry was utilized to investigate the growth kinetics and surface morphology in canavalin, thaumatin, and turnip yellow mosaic virus (TYMV) crystallization. Interferometric patterns and kinetic measurements from growing macromolecular crystals as small as 20 p~m were obtained. This study shows that for the crystallization of canavalin, dislocations are the sources of growth steps on the surfaces of growing crystals. Supersaturation dependencies of the normal growth rates, tangential growth step velocities, and the slopes of the dislocation hillocks were determined. The kinetic coefficient 13 was estimated for canavalin grown from two different precipitant systems to be 3.2 × 10 - 4 and 5.3 × 10 - 4 c m s - 1 , respectively. The change in activities of dislocation sources under different growth conditions was analyzed.

I. I n t r o d u c t i o n

Crystallization of biological macromolecules has become the rate-limiting step in most X-ray structure analyses, since little is known about the mechanisms of nucleation, crystallization, and defect formation; and virtually nothing is known of the fundamental thermodynamic and kinetic parameters that govern macromolecular crystallization. Undoubtedly, a more-comprehensive understanding of all of these questions will be required in order to grow large, high-quality macromolecular crystals and to fulfil the potential of X-ray crystallography for molecular biology and medicine. Michelson interferometry is a powerful in situ technique for both the qualitative characterization of surface morphology, crystal growth, and the precise quantization of parameters that govern the process. It

* Corresponding author. Fax: + 1 909 787 3590.

has been successfully applied to the investigation of solution-crystal-growth kinetics for a number of different inorganic [1-4] and organic [5] systems. Michelson interferometry was also recently applied to investigate the crystallization of lysozyme [6,7].

2. M a t e r i a l s

Canavalin (molecular weight M r = 147000 Da) was purified from jack bean (Canavalis ensiformis) meal (Sigma Biochemical Co.) as described in Ref. [8]. Rhombohedral crystalsoof canavalin (space group R3 with a = b = c = 83 A and ~ = 111.1 ° (equivalent tril~ly centered hexagonal cell a = b = 126 A, c = 51 A [9]) were grown by a batch method of mixing 1 5 - 5 0 mg m1-1 protein dissolved in water with the equal amount of precipitant (2 × Dullecco's phosphate-buffered saline (DPBS) buffered at pH 6.8 or 20% 2-methyl-2,4-pentanediol (MPD), buffered at pH 8.0).

0022-0248/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved SSDI 0022-0248(95)00554-4

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The turnip yellow mosaic virus (TYMV) (M r 5.8 X 10 6 Da) was purified from infected 6 - 8 week

old Chinese cabbage leaves as described in [10]. The hexagonal bipyramidal crystals oOf TYMV °(space group P6222 with a = b = 525 A, c = 315 A [10]) crystals were grown by a batch method of mixing 35 mg ml -I virus solutions with 1.6-1.65M ammonium phosphate. TYMV is a T = 3 virus having a unipar~t,ite genome and a particle diameter of about 280 A, making it the largest and most-complex particle whose crystallization behavior has been seriously investigated. Thaumatin (M r = 22000 Da) from the arils of the African shrub Thaumatoccus daniellii was purchased from Sigma Biochemical Co. Tetragonal bipyramidal thaumatin crystals (space group P41212 with a = b = 58.6 and c = 157.8 A [11]) were grown by a batch method of mixing 40 mg ml-1 of protein dissolved in water with 1.4M sodium-potassium tartrate b u f f e r e d by 0 . 1 M ADA (sodium-N-2acetamidoiminodiacetic acid) at pH 6.5. The macromolecules investigated in this work all represent excellent systems for crystallization studies because they are extremely stable, and large, reproducible crystals can be grown over a wide temperature range within a few hours.

3. Methods

Michelson interferometry was utilized to observe directly dislocation sources of step generation on the surfaces of growing crystals and to measure the normal growth rates and the slopes of the dislocation hillocks. It was used as well to calculate the tangential growth rates of the steps. These, in turn, allowed a determination of the kinetic coefficient [3. A detailed description of the experimental apparatus has been presented in Ref. [12]. The major difficulty in investigating macromolecular crystallization using interferometric techniques is the small size of the crystals which rarely exceed a millimeter in size. Thus high magnification was obligitory in the experimental arrangement, and this allowed us to obtain interferometric patterns from macromolecular crystals as small as 20 txm. Because the macromolecular systems investigated in this work did not have particularly strong tempera-

ture dependencies of solubility, the supersaturation dependencies of the crystal growth parameters were obtained by altering the initial protein concentrations. Crystals were nucleated in a small droplet within the growth cell having a volume of 3 ~1, and then, the entire volume of the cell of 60 Ixl was filled with a mixture of protein and precipitant at appropriate concentrations. This experimental approach appears to be very useful because it allows us to separate nucleation from the growth phase of crystallization. Thus, for example, canavalin crystals were nucleated by batch procedures at initial protein concentrations of 15 mg ml -l and higher. These seed crystals continued to grow, however, at protein concentrations as low as 5 mg m1-1, clearly under metastable conditions. This is significant, because in most macromolecular crystallization techniques, crystal growth proceeds at the extreme supersaturations required for nucleation, often resulting in poor crystal quality. In all the experiments, there were no more then 2 - 5 small crystals in the growth cell. Thus, in the initial stages of crystallization, which lasted in many experiments for up to 40-50 h [12], the supersaturation was stable, with normal growth rates virtually constant. Ultimately, the normal growth rate R decreased in time because of protein solution depletion. All measurements of crystallization parameters for specific supersaturations were made during crystallization periods when normal growth rates were virtually constant. The solution supersaturation tr was determined as = ln(c/Ce), where c and c e are the macromolecule concentration and solubility, respectively. Solubilities c e were measured as macromolecule concentrations of the supernatant solutions in the growth cells when the crystal growth was completed, usually after 2 - 3 months.

4. Results and discussion 4.1. Canavalin crystallization 4.1.1. Fluctuations in normal growth rates due to changes in activities o f dislocation sources In all of our experiments, dislocation sources were observed on the surfaces of growing crystals, and these sources dominated growth. These disloca-

Yu.G. Kuznetsov et al. / Journal of Crystal Growth 166 (1996) 913-918

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Fig. 1. Changes in the activities of dislocation sources in canavalin crystallization.

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tion sources produced rather steep dislocation hillocks with slopes ranging from 5 X 10 -3 to 1.3 × 10 2. The values for the slopes of the dislocation hillocks were virtually the same over the entire supersaturation range investigated, which indicates [1,2] that they were produced by strong, complex dislocation sources.

Canavalin crystal faces typically had several growth dislocation hillocks. During crystallization at constant supersaturation, the observed fluctuations in normal growth rates were principally due to changes in the activities of the dislocation sources. We noted, during the periods when kinetic measurements were being made, that different dislocation sources of similar activities (equal slopes of dislocation hillocks) produced similar values for normal and tangential growth rates, further indicating the stability of the supersaturation conditions. Typical examples of changes in the activities of dislocation sources are illustrated in Fig. 1. In Fig. la, the entire face is dominated by a single steep growth hillock, with a slope p = 7 × 10 -3, formed by a strong dislocation source in the center of the face. The normal growth rate of the crystalline face was 1.4 × 10 -7 c m s -1. Strong, complex dislocation sources, always form from a set of single dislocations, and the distance between the points of their emergence on a growing face can change upon the deposition of new layers [1,2]. These reduce the activities of initial, complex dislocation sources thereby resulting in the appearance of new dislocation sources. This is illustrated in Figs. l b - l e . When the activity of a dominant dislocation source decreased, a number of weaker dislocation sources emerged on the growing surface of the crystal. These produced growth hillocks with slopes and normal growth rates that decreased correspondingly to 3.4 × 10 -3 and 5.7 × 10 -8 cm s -1. A stronger dislocation source, having a hillock slope and normal growth rate similar to the dislocation source described above (Fig. 1, frame a), later appeared on the face and began to dominate face growth (Fig. 1, frames f-h). The fluctuations of normal growth rates, due to change in the activities of dislocation sources, were more prominent at higher supersaturation, while at lower solution supersaturations the same dislocation source was generally responsible for growth steps during the entire course of an experiment.

4.1.2. Growth kinetics The kinetics of canavalin crystallization was investigated under two different sets of conditions. These involved, in one case, application of pH dependent (2 × DPBS, pH = 6.8) and organic (20% MPD, pH = 8.0) precipitants. Normal growth rates of canavalin crystals (Fig. 2) and tangential step velocities (Fig. 3) were measured in the supersaturation ranges ~r = 0.63-2.3 (2 × DPBS, pH = 6.8) and c r = 0 . 1 8 - 2 . 3 2 (20% MPD, p H = 8 . 0 ) . Normal growth rates were typically measured for 2 - 3 crystals at the same solution supersaturation. In all of our experiments, because strong dislocation sources produced growth steps (Section 4.1.1), normal growth rates and dislocation hillock slopes were reproducible. Under these supersaturation conditions, the normal growth rate R varied from only 6 × 10-10 to 1.7 × 10 -7 cm s - ] (Fig. 2). This corresponds to the deposition of one molecular layer in 20 min, and in 4 s, respectively. A normal growth rate R of 6 × 10-10 cm s -1 was measured for 4 days at a solution supersaturation er = 0.18 (since for small or, l n ( c / c e) ~- (c - c e) × lO0/ce (in %), a traditional form for the supersaturation expression in conventional molecules solution crystal growth, thus ~r = 20%). This represents an unusually low supersaturation value, as it is commonly assumed that a supersaturation of several hundred percent is required for macromolecular crystal growth to occur. We noted,

20-

0

0

1 Z SUP~RSATU~TIO~ O

3

Fig. 2. Supersaturation d e p e n d e n c i e s o f the normal growth rate R for canavalin crystallization: canavalin incubated with 1 - 2 × DPBS, pH = 6.8; 2 - 2 0 % MPD, pH = 6.8.

Yu. G. Kuznetsov et al. / Journal of Crystal Growth 166 (1996) 913-918 15

10 1 o

Z

0' 0

4

6

8

(C - Ce) / Ce

Fig. 3. Supersaturation dependencies of tangential step rates for canavalin crystallization: canavalin incubated with 1 - 2 X D P B S , pH = 6,8; 2-20% MPD, pH = 6.8.

however, that at supersaturations lower then (r = 0.18 canavalin crystal growth did not initiate for a period of 4 - 5 days. Tangential step velocities c varied in the range of 8 X 10-8-1.1 X 10 -5 cm s - 1 (for 20% MPD, pH = 6.8) and 2 . 2 X 1 0 - 6 - 1 . 3 6 X 10 -5 cm s -1 (for 2 X DPBS, pH = 6.8). Dependencies for tangential step velocities versus s - - ( c - c e ) / c e are presented in Fig. 3. From such experimental data, the kinetic coefficient 13 (rate of incorporation of canavalin molecules in the growing steps) can be estimated. The kinetic coefficient 13 is defined [13] by the relationship v = f113(c - ce) ~13CeS , where 12 = 4.1 X 10 - 1 9 c m 3 is the specific volume of the canavalin molecule in the crystal with c and c c being the initial and equilibrium volume concentrations of the dissolved canavalin, respectively. It can be seen in Fig. 3, that in the case where 2 x DPBS was utilized as precipitant, the dependency v(s) is linear, extrapolating to the origin. The kinetic coefficient 13 was estimated here to be 5.3 X 10 -4 cm s - j . In the case where 20% MPD was used as precipitant (Fig. 3, line 2), a deviation of linearity of v(s) at low supersaturation was observed. From the linear portion of the v(s) curve at s greater than 3.5, the kinetic coefficient 13 was estimated to be 3.2 X 10 -4 cm s-~ These kinds of nonlinear supersaturation dependencies for growth-step velocities have been observed numerous times for small-molecule inorganic crystals grown from solution [1-5]. In all =

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cases, the nonlinearities were ascribed to the effects of impurities. At lower supersaturation, a longer time t = h / R is required to form an elementary layer with height h on the surface of a growing crystal. Thus, impurities adsorb on the terraces between steps and decrease the rate of step advancement. At higher supersaturation, step velocities increase and reduce the amounts of impurities adsorbed on the terraces, thereby resulting in an increase in step velocities. Under conditions of low supersaturation, as noted above, crystals continued to grow with a rate of 6 x 10 -1° cm s - j . It appears that for canavalin crystallization, therefore, no pronounced "dead zone" exists. Impurities appear to adsorb on terraces in insufficient amounts to produce the cessation of step movement. This, probably indicates that mobilities of relevant impurities must be no more then those for the canavalin molecules themselves. This suggests that the relevant impurities are likely to be rather large particles. Impurities, however, which cause retardation of growth steps at low supersaturation and those responsible for a "dead zone" could be quite different [14]. 4.2. TYMV and thaumatin crystallization TYMV and thaumatin crystallization, currently under additional study, have not yet been investigated over as wide a supersaturation range as canavalin. At the solution supersaturation ~r = 1.05, TYMV crystals grew with a normal growth rate R of 4 X 10 -8 cm s -~, corresponding to the deposition of one molecular layer in about 70 s. Steep hillocks were observed on the surfaces of TYMV crystals. Their slopes, varied in the range of (1.1-1.6) X 10 -2. Tangential-step growth rates varied in the range (2.5-3.7) × 10 6 c m s - I , Thaumatin crystallization was investigated at a solution supersaturation cr = 1.8. The normal and tangential growth rate R were equal to 8 X 10 - 9 and 1.9 X 10 - 6 c m S - I , respectively.

Acknowledgements This work was supported by grants from the National Aeronautics and Space Administration. We thank C. Leja for preparing the canavalin.

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[7] S. Miyashita, H. Komatsu, Y. Suzuki and T. Nakada, J. Crystal Growth 141 (1994) 419. [8] J.B. Sumner and S.F. Howell, J. Biol. Chem. 113 (1936) 607. [9] T.P. Ko, J.D. Ng, J. Day, A. Greenwood and A. McPherson, Acta Cryst. D 49 (1993) 478. [10] M. Canady, J. Day and A. McPherson, Proteins 21 (1995) 78. [11] T.P. Ko, J. Day, A. Greenwood and A. McPherson, Acta Cryst. D 50 (1994) 813. [12] Yu.G. Kuznetsov, A.J. Malkin, A. Greenwood and A. McPherson, J. Struct. Biol. 114 (1995) 184. [13] A.A. Chernov, Modern Crystallography III, Crystal Growth (Springer, Berlin, 1984). [14] A.A. Chernov and A.J. Malkin, J. Crystal Growth 92 (1988) 432.