- Email: [email protected]
Protein crystal growth rates are face-specifically modified by structurally related contaminants
,. . . . . . . .
CRYSTAL G R O W T H
ELSEVIER
Journal of Crystal Growth 171 (1997) 559-565
Protein crystal growth rates are face-specifically modified by structurally related contaminants Joachim Hirschler a,b, Juan Carlos Fontecilla-Camps a,* a Laboratoire de Cristallographie et de Cristallog~nbse des Prot6ines, lnstitut de Biologie Structurale Jean-Pierre Ebel, CEA-CNRS, 41, AL enue des Martyrs, F-38027 Grenoble Cedex 1, France b A~rospatiale E~pace et D#fense, 66, Route de Verneuil, BP2, F-78133 Les Mureaux Cedex, France Received 1 March 1996; accepted 20 June 1996
Abstract
Growth rates of turkey egg-white lysozyme (TEWL) crystal faces have been measured in uncontaminated solutions as well as in solutions contaminated by the homologous hen egg-white lysozyme (HEWL). Comparison of growth rates from uncontaminated and contaminated solutions shows that the growth rate of the {112} faces drops significantly in the presence of the contaminant, whereas the growth rate of the {ll0} faces does not change. This demonstrates that HEWL acts specifically on the growth process of the {112} faces.
1. Introduction
There is little need to stress the importance of obtaining well-diffracting protein crystals for structural studies. The purity of the protein is one of the key parameters determining the crystallization success (e.g. Ref. [1]). Yet protein solutions which are thought to be homogenous still contain considerable amounts of contaminants (e.g. Ref. [2]). The influence of contaminants on the protein crystal growth process is therefore of considerable interest (e.g. Ref. [3]). Previous studies from our laboratory have shown that it is not the overall presence of a contaminant, but also its relatedness to the protein to be crystallized, which influences the growth process. We have crystallized turkey egg-white lysozyme (TEWL) con-
* Corresponding [email protected].
author.
Fax:
+33
76885122;
E-mail:
taminated by either hen egg-white lysozyme (HEWL) or ribonuclease A [4,5]. HEWL and TEWL are homologous proteins. Only seven out of their 129 residues are different and their three-dimensional structures are very similar (Protein Data Bank entries 61yz and 21z2). Ribonuclease A is a different protein with no structural resemblance to lysozyme, though it has similar molecular weight and isoelectric point. HEWL contamination of TEWL solutions changed the aspect ratio of the resulting crystals towards lower length-to-width ratios (see Fig. 1). The cell parameters and the symmetry of the crystals grown in contaminated solution were the same as those of uncontaminated TEWL crystals (see Section 2). Yet significant amounts of the HEWL contaminant seem to be included in these crystals, approximately proportional to the initial contaminant concentration of the growth solution (see also Ref. [5]). Contamination of TEWL by ribonuclease A, on the other hand,
0022-0248/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S 0 0 2 2 - 0 2 4 8 ( 9 6 ) 0 0 6 8 8 - 4
J. Hirschler, J.C. Fontecilla-Camps / Journal of Co,stal Growth 171 (1997) 559-565
560
{I 12} fsee
{I I0} face
Fig. 1. Schematic drawing of a T E W L crystal (space group P6122). The crystal rests on a {110} face in the growth cell. Co-crystallization of H E W L leads to crystals which are shortened along the c-axis.
did not modify crystal morphology. This shows the importance of the contaminant's structural resemblance to the principal compound in order to interact in the crystal growth process. We proposed that the contaminant's influence is face-specific and that its effect can be compared to what is known from crystal growth of small organic molecules in the presence of tailor-made additives [6]. According to this model, additives with a structure similar to that of the main compound may act on only one set of crystal faces. We speculated that HEWL interacts preferentially with the apical {112} faces of the TEWL crystal, but only to a lesser degree (or not at all) with the lateral {110} faces. This specific interaction would significantly slow down the crystal growth rates of the {112} faces, but not those of the {110} faces. In order to verify our hypothesis, we have carried out a series of face growth rate measurements on TEWL crystal in both uncontaminated solution and in solution contaminated by HEWL. To our knowledge this is the first report on protein crystal growth rates in the presence of homologous contaminants.
tion window and the upper plate are then tightened to the center plate to close the cell and assure air-tightness. Crystal growth is monitored via the two observation windows. With our growth cell design, the protein growth solution cannot be exchanged during the course of an experiment. Consequently, the growing crystal will decrease the overall protein concentration, which, in turn, will reduce the crystal growth rates. We arbitrarily decided that this growth rate reduction should not exceed 7%. Using Eq. (1) with a supersaturation O" = ( C T E w L -- C s o l ) / C s o I = 8.38 (CTEwL i s t h e TEWL concentration, C~o1 is the TEWL solubility of 1.6 m g / m l , see below for exact conditions) corresponding to 15 m g / m l TEWL and a value for r of 2 (see Section 3) shows that a supersaturation decrease of approximately 3% will decrease the growth rate by no more than 6.6%. The calculation of the minimum growth cell volume needed in order not to surpass this 3% decrease is based on the previously published TEWL crystallization conditions by the vapor-diffusion method [4]. A typical crystal obtained in a drop of 4 ILl volume at 15 m g / m l initial TEWL concentration stops growing at a final TEWL concentration of 1.6 m g / m l (see below). This decrease indicates that about 0.054 mg TEWL have been incorporated into the crystal. As this crystal is much larger ( ~ 0.7 mm longest dimension) than the crystals used for our growth rate measurements (see below), this corresponds to the maximum foreseeable TEWL loss in the growth chamber. This chamber has a diameter of 8.5 mm and a height of 4 mm (see 8,5 mm i
.
.
.
i
.
. . . . . . . . . . . .
2. Experimental procedure The crystal growth cells we used are based on a design of Pusey ([7], see Fig. 2). The lower cell plate is first assembled along with the lower observation window and the center plate. This gives a water-tight cavity, into which the protein growth solution is poured. A previously obtained seed crystal is then transferred to this growth solution, where it rests on the lower observation window. The upper observa-
4 mm -"" " [ ' ~ . ~
[ : ~ 1
upper cover slip center plate lower cover slip
Fig. 2. Explosion drawing of the growth cell. Upper, center and lower plates are made of acrylic plastic. The observation windows are the cover slips used on Linbro Plates. The growth chamber is formed by the hole in the center plate and by the two observation windows. The tightening screws have been omitted in the figure for simplicity.
561
J. Hirwhler, J.C. Fontecilla-Camps / Journal of Crystal Growth 171 ( 19971 559-565
Fig. 2), which corresponds to a volume of 227 Ixl. When completely filled with a 15 m g / m l TEWL growth solution, this chamber contains 3.0 mg of available TEWL. The maximum loss of about 0.054 mg TEWL by the crystal growth will thus decrease the initial TEWL concentration by only 1.8%. Even at 10 m g / m l TEWL, this decrease would only amount to 2.8%, which is within the set limits. The growth chamber is also large enough to avoid obstructing convective flow around the growing crystal. This problem may be encountered in a more constrained space, which may reduce transport kinetics
[8]. The TEWL seed crystals were obtained by the vapor-diffusion method at 20°C in sitting drops consisting of a mixture of 2 b~l of 30 m g / m l commercial-grade TEWL (Sigma, crystallized, dialyzed and lyophilized) and of 2 ILl precipitant solution on flat insets [9] in Linbro Plates [10]. The precipitant solution at pH 5.8 consisted of 2.40M NaC1, a 0.1M HEPES buffer (N-[2-hydroxyethyl]piperazine-N'-[2ethanesulfonic acid], Fluka BioChimica) and 15raM NaN 3 used as an antibacterial agent. A reservoir volume of 1 ml of the precipitant solution was used. After equilibration of the drop against the reservoir, (expected final drop volume of 2 b~l, corresponding approximately to initial concentrations of 2.40M NaC1, 0.1M HEPES and 15mM NaN 3 at pH 5.8), the solubility of TEWL crystals was determined to be 1.6 m g / m l [4]. These conditions typically yield 1 to 5 well-shaped crystals of up to 0.7 mm in their longest dimension.
Their cell parameters are a = b = 73.2 ,~ c = 86.8 A, a =/3 = 90.0 ° y = 120.0, space group P6~22 [4]. Occasional appearance of polycrystalline aggregates could not be suppressed either by filtering or dialysis of the protein solution. The seed crystals were slowly conditioned to the growth cell solution by repeated addition of small aliquots of this solution to the drop containing the seed crystal (three times during several hours for TEWL concentrations of 15 mg/ml). This pre-equilibration was needed to minimize the shock of transferring the crystals from the vapor-diffusion drop to the growth cell experiment (marked as " A " in Table 1). Crystals that had grown too large (larger than 300 ~m in their longest dimension) were partially dissolved by diluting the corresponding reservoir solutions ( " B " in Table 1). Under these conditions, the crystals shrank to sizes below 250 ~m over a few days. The rough surfaces they had acquired during this process became smooth again soon after they were transferred to the growth solution. Crystals which were directly transferred to the growth cell without pre-equilibration were also tested ( " C " in Table 11. The cell growth solution consisted of a one-to-one mixture of the precipitant solution described above and of TEWL, with or without 33% HEWL contaminant ( w / w of total protein), dissolved in water. HEWL was added so that the TEWL concentrations stayed constant at their respective levels of 10, 15 or 20 m g / m l (corresponding to o- = 5.25, 8.38 or 11.5, respectively, not taking into account the possible influence of HEWL on TEWL solubility). TEWL
Table 1 TEWL crystal growth rates G for the {112} and {110} faces at 15 m g / m l TEWL in uncontaminated solution Crystal parameters
Growth rates G and correlation coefficients R 2
Initial length (txm)
Initial width (ixm)
Pretreatment
Experiment duration (h)
G{ 112} (tzm/h)
R 2 of G{112}
G{ 110} (Ixm/h)
R 2 of
73 93 133 213 240 253
40 60 73 89 107 80
A A B C B B
53 70 34 22 45 69
4.54 5.41 5.65 3.92 4.83 5.45
1.000 0.999 0.999 0.995 0.999 0.995
0.201 0.287 0.208 0.303 (I.340 (I.275
0.944 0.964 0.812 0.934 0.985 0.904
G{110}
The effect of different initial sizes and pre-treatments on growth rate measurements was verified. Neither the linearity of the individual face growth data over time (R 2) nor the obtained growth rates G showed any systematic variation. The data are plotted in Fig. 3. A: seed crystal adapted to growth cell conditions before its transfer. B: seed crystal larger than 300 ixm in length partially dissolved, and then transferred. C: seed crystal transferred to the growth cell without adaptation.
J. Hirschler, J. C. Fontecilla-Camps / Journal of Crystal Growth 171 (1997)559-565
562
i
400
was used as purchased from Sigma, and HEWL was filtered three times over 3 kDa filter membranes (Microsep 3K for 8 h at 6000g). The growth cells were stored in insulating vessels and kept at 20 _+ 0.5°C. At regular intervals they were removed from the vessels and the length and width of the crystals were measured under a dissecting microscope. An experiment was stopped when extensive secondary nucleation was detected, as this would decrease the protein concentration beyond the set limits. Crystal dimensions were measured from edge to edge and then converted to obtain the orthogonal dimensions from crystal face to crystal face. Known interfacial angles of 60 ° between adjacent {110} faces and of 124 ° between {110} and {112} faces were used for this conversion [4]. These measurements were plotted against time, and the data were approximated to a straight line by the least squares method. The data quality was assessed by the correlation coefficient R 2 of this straight line, whose slope corresponds to the face growth rate G. A first series of experiments was carried out to assess the possible influence of different initial crystal sizes on the growth rates [11] a n d / o r the possible effects due to different pre-treatmerits of the seed crystals.
" • 350 t: o L
250
~
-
i
i
L
i
,~
15 mg/ml T E W L
+ +
uncontarni rmted • {112}
4:
o {110) HEWL-contamin. + {112} x {110}
~A
•
+ +
•A
•
200 150
~.~
tt I00
50
++
~A +
~ ~4:'~i• + .
20
40
60
time
80
1 O0
120
[hours]
Fig. 3. T E W L crystal face growth over time in uncontaminated and in HEWL-contaminated T E W L solution at 15 m g / m l TEWL. The data are summarized in Table 2. In HEWL-contaminated solutions, the {I 12} face growth rates are slowed down, whereas the {110} face growth rates are not significantly changed.
and somewhat lower for data of the lateral faces {110} (abbreviated as G{110}, 0.812 < R 2 < 0.985). Fig. 1 shows the crystal faces and their indices. The observed growth rates lie all within the same range for a given set of faces. This agreement is better for G{112} than for G{110}. This can be seen by calculating the ratio of the root mean square deviations (rmsd) of G to the respective value of G: G{112} is 4.97 t x m / h with a rmsd of 0.662 (0.662/4.97 = 0.133), whereas G{110} is 0.269 ~ m / h with a rmsd of 0.0546 (0.0546/0.269 = 0.202, see Table 2). Neither different initial sizes nor different pre-treatments
3. Results and discussion The validation experiments of the growth cell in contaminant-free 15 m g / m l TEWL solution show that linear face growth rate data can be obtained with high correlation coefficients R 2 (Table 1 and Fig. 3). R 2 is high for growth rate data of the apical faces {112} (abbreviated as G{112}, 0.995 < R 2 < 1 . 0 0 0 ) , Table 2
T E W L crystal growth rates (G) of the apical {112} laces and the lateral {110} faces in uncontaminated and HEWL-contaminated solutions (33% H E W L ( w / w total protein)); root mean square deviations (rmsd) are calculated from repeated experiments
Uncontaminated
Contaminated
CTEWL (mg/ml)
G{112} (~zm/h)
rmsd of G{112}
G{I t0} (I-~m/h)
rmsd of G{110}
Supersaturation cr
G{112}/ G{110}
10 15 20 10 15
0.588 4.97 18.5 0.486 3.02
0.0479 0.662 1.65 0.0438 0.213
-
0.0546 0.672 0.0798
5.25 8.38 11.5 5.25 b 8.38 b
18.5 12.2 _ 10.8
'~ 0.269 1.52 - ~ 0.279
" Growth rates were too low to be determined. b Supersaturation ~r of TEWL, without taking into account a possible influence of H E W L on T E W L solubility.
J. Hirschler, J.C. Fontecilla-Camps / Journal of Co'stal Growth 171 (1997) 559-565
of the seed crystals had any systematic effect o n R 2 of the individual measurements or on the averaged growth rates G. The data presented here show that a structurally related protein contaminant can selectively modify the face growth kinetics of a protein crystal. Contamination of a 15 m g / m l TEWL solution by 7.5 m g / m l HEWL did not significantly modify the face growth rates of the lateral {110} faces of the TEWL crystal (0.279 compared to 0.269 I~m/h in uncontaminated solution), whereas it did reduce those of the apical {112} faces by 39% (3.02 compared to 4.97 ~ m / h , Table 2). R 2 is comparable with those obtained from measurements in uncontaminated solutions (not shown). The ratios of rmsd(G) to G are higher for G{II0} than for G{112}, as already observed in uncontaminated solutions. This specific influence of crystal face growth kinetics shows that the morphological changes previously observed in HEWL-contaminated crystallization of TEWL crystals [4,5,12] are indeed kinetic phenomena. Briefly, in our model we attribute these changes to the structural relationship between TEWL and HEWL, which may exert different interactions
L
~
i
20 mglmlTEWL
90
• ( I 12}, uncontaminated TEWL o { 1 10}, uncontarninated TEWL
8o
e.
70
0 I,.
60
(j
50
t
- =°1 ;o'd-;.,; mO J ~J ~4001 TEWL
At 4O 30
20-
"
:°o°ot
e 2
0
L SO
I00
1
10
4- (I 12) HEWL-oontarnin. [
I~
200
2~
300
3~
time [hours]
AA
o ( oi~I~.
oo8O -
,
4
.
,
8
,
,
.
~
•
,
12
16
20
time
[hours]
,
,
24
2'8
Fig. 4. Face growth data observed with 20 m g / m l uncontaminated TEWL and 10 m g / m l contaminated TEWL solutions (inset). The data show the strong dependence on the TEWL concentration. At 10 m g / m l TEWL, the contaminant reduces the {112} growth rates less than it does at 15 m g / m l (see Fig. 3). {110} growth rates at 10 m g / m l TEWL could not be measured.
563
with the various faces of the growing TEWL crystal [4]. The {112} faces display more affinity for the contaminant than the {110} faces. Thus the {112} faces are less accessible to TEWL molecules due to their higher partial contaminant coverage. Their growth will then be slower than that of the {110} faces, as we have shown with our measurements. This represents, according to our knowledge, the first report on face growth rates of protein crystals in a contaminated solution. Five m g / m l HEWL contamination of a 10 m g / m l TEWL solution reduces the growth rates of the {112} faces by 17% (from 0.588 to 0.486 tzm/h, Table 2 and Fig. 4). {110} face growth rates were too low to be reliably measured (see below). Consequently, we do not know the contaminant's effects on the {110} faces at this TEWL concentration. Yet from the results at 15 m g / m l TEWL we have seen that the {110} face growth rates were not significantly influenced by the contaminant. By extension, we can assume equally unmodified {110} face growth rates at 10 m g / m l TEWL. Thus the G{112} decrease of 17% at 10 m g / m l TEWL compares to the 39% decrease at 15 m g / m l TEWL. This indicates an increasing effect of the contaminant with increasing supersaturation. If confirmed by further measurements, this supersaturation dependence may be opposed to that of crystal growth of contaminated low molecular weight materials. In fact, according to a recent model by Kubota and Mullin, the lower the supersaturation of the principal compound, the more pronounced the effect of the contaminant is [13]. Yet the comparability between the two domains of crystal growth is limited. Firstly, the supersaturation range employed in protein crystal growth is orders of magnitude higher than that found in the crystal growth of low molecular weight materials. Secondly, the respective crystallizing species may be differently aggregated: they are assumed to be monomers in the case of low molecular weight materials, whereas proteins may exist as larger aggregates. The aggregates' sizes may furthermore change with supersaturation [8,14,15]. The uncontaminated TEWL growth rates observed here at 15 mg/ml TEWL are of the same order of magnitude as those published for HEWL [16]. However, in our case the variation of the growth rates as a function of supersaturation seems
564
J. Hirschler, J.C. Fontecilla-Camps / Journal of Crystal Growth 171 (1997) 559-565
to be different (Table 2 and below). The empirical equation
G = k o r,
(l)
is commonly used to fit the dependence of the growth rate on the supersaturation (growth rate G, supersaturation o', constant k). The slope of a log(o-)-log(G) plot of TEWL growth rates gives for r a value of 4.4 (R 2 = 0.999). The corresponding value for HEWL growth rates is r = 2.0 [17]. The value of r = 2 is generally taken to indicate a spiral growth mechanism, whereas r > 2 indicates growth via surface nucleation [18]. Thus HEWL and TEWL crystallization under the respective conditions may proceed via different growth mechanisms. However, this approach can only serve as a first indicator of the growth mechanism, as only techniques such as atomic force microscopy or interferometry can yield information on a molecular level. With a factor r of 4.4, we also have to modify our initial assumption for the cell volume calculation based on r = 2 (see Section 2). It actually indicates a maximum error in G of 12% instead of the previous 6.6%. However, as our assumption takes into account a far larger crystal than those crystals measured here, the actual deviation in G will have been smaller than 12%. The ratio G{ 112}/G{ 110} in uncontaminated solution is 18.5 and 12.2 at 15 and 20 m g / m l TEWL, respectively (Table 2). At 10 m g / m l TEWL, G{II2} could be reliably measured for up to 340 h, whereas the G{ll0} were too low to be measured. These ratios show that the different supersaturation levels influence the two face growth rates (G{II2} and G{110}) differently, as it was also observed on tetragonal HEWL crystals [19]. This effect may be explained by different absorption of intrinsic impurities on the crystal faces [20]. According to a recent report, crystal-impurity interactions can also depend on the crystal size [3]: crystals with maximum lengths of under 40 ~m were shown to include higher levels of NaC1 and inherent protein impurities than larger crystals, possibly infringing crystal growth. Yet this does not pertain our experiments, as our smallest crystal was twice as long. We altered the TEWL supersaturation by varying the TEWL concentration in our experiments. Another possibility is to vary protein solubility using
temperature (see Ref. [16] for measurements of HEWL growth rates). We chose not to use this method, since the kinetics of every other temperature-dependent process during crystallization may also be modified [20].
4. Conclusion This first determination of crystal growth rates in contaminated protein solution confirms that structurally related protein contaminants influence growth rates of protein crystals in a face-specific manner. Co-crystallization of the contaminant HEWL with TEWL leads to a significant drop of growth rates of the apical {112} faces of the crystal, but does not influence the growth rates of the lateral {110} faces. These results on contaminated protein crystal growth may be explained by what is known concerning the face-specific influence of structurally related contaminants in organic crystal growth. Together with more detailed knowledge on molecular-level protein contaminant-protein crystal interactions, crystal engineering may thus become a feasible means to modify protein crystal morphology systematically for research or for industrial applications. Further measurements should validate our preliminary results concerning the contaminant's supersaturation dependence. If so, it may be advantageous to crystallize proteins in a supersaturation range where the deleterious effects of contaminants, inherent to protein crystallization, are minimized.
Acknowledgements We thank Dr. Pusey for providing help on the initial design of our growth cell and Dr. Sunawaga for pointing out that "aspect ratio" is the appropriate expression to use for evaluating crystal morphology.
References [1] R. Gieg6, B. Lorber and A. Theobald-Dietrich, Acta Cryst. D 50 (1994) 339. [2] P.G. Vekilov, M. Ataka and T. Katsura, Acta Cryst. D 51 (1995) 207.
J. Hirschler, J.C. Fontecilla-Camps / Journal of Cr3'stal Growth 171 (1997)559-565 [3] P.G. Vekilov, L.A. Monaco, B.R. Thomas, V. Stojanoff and F. Rosenberger, Acta Cryst. D, submitted. [4] J. Hirschler and J.C. Fontecilla-Camps, Acta Cryst. D 52 (1996) 806. [5] C. Abergel, M.P. Nesa and J.C. Fontecilla-Camps, J. Crystal Growth 110 (1991) 11. [6] M. Lahav and L. Leiserowitz, J. Phys. D 26 (1993) B22. [7] M.L. Pusey, Anal. Biochem. 158 (1986) 50. [8] S. Miyashita, H. Komatsu, Y. Suzuki and T. Nakada, J. Crystal Growth 141 (1994) 419. [9] T.M.B. Soriano and J.C. Fontecilla-Camps, J. Appl. Cryst. 26 (1993) 558. [10] J. Hirschler, M.H. Charon and J.C. Fontecilla-Camps, Prot. Sci. 4 (1995) 2573. [1 I] M.L. Pusey and R. Naumann, J. Crystal Growth 76 (1986) 593.
565
[12] K. Provost, M.C. Robert, J. Crystal Growth 156 (1995) 112. [13] N. Kubota and J.W. Mullin, J. Crystal Growth 152 (1995) 203. [14] M. Li, A. Nadarajah and M.L. Pusey, J. Crystal Growth 156 (1995) 121. [15] F. Rosenberger, P.G. Vekilov, M. Muschol and B.R. Thomas, J. Crystal Growth 168 (1996) I. [16] E. Forsythe and M.L. Pusey, J. Crystal Growth 139 (1994) 89. [17] M.L. Pusey, R.S. Snyder and R. Naumann, J. Biol. Chem. 261 (1986) 6524. [18] A.E. Nielsen, J. Crystal Growth 67 (1984) 289. [19] S.D. Durbin and G. Feher, J. Crystal Growth 76 (1986) 583. [20] L.A. Monaco and F. Rosenberger, J. Crystal Growth 129 (1993) 465.
CRYSTAL G R O W T H
ELSEVIER
Journal of Crystal Growth 171 (1997) 559-565
Protein crystal growth rates are face-specifically modified by structurally related contaminants Joachim Hirschler a,b, Juan Carlos Fontecilla-Camps a,* a Laboratoire de Cristallographie et de Cristallog~nbse des Prot6ines, lnstitut de Biologie Structurale Jean-Pierre Ebel, CEA-CNRS, 41, AL enue des Martyrs, F-38027 Grenoble Cedex 1, France b A~rospatiale E~pace et D#fense, 66, Route de Verneuil, BP2, F-78133 Les Mureaux Cedex, France Received 1 March 1996; accepted 20 June 1996
Abstract
Growth rates of turkey egg-white lysozyme (TEWL) crystal faces have been measured in uncontaminated solutions as well as in solutions contaminated by the homologous hen egg-white lysozyme (HEWL). Comparison of growth rates from uncontaminated and contaminated solutions shows that the growth rate of the {112} faces drops significantly in the presence of the contaminant, whereas the growth rate of the {ll0} faces does not change. This demonstrates that HEWL acts specifically on the growth process of the {112} faces.
1. Introduction
There is little need to stress the importance of obtaining well-diffracting protein crystals for structural studies. The purity of the protein is one of the key parameters determining the crystallization success (e.g. Ref. [1]). Yet protein solutions which are thought to be homogenous still contain considerable amounts of contaminants (e.g. Ref. [2]). The influence of contaminants on the protein crystal growth process is therefore of considerable interest (e.g. Ref. [3]). Previous studies from our laboratory have shown that it is not the overall presence of a contaminant, but also its relatedness to the protein to be crystallized, which influences the growth process. We have crystallized turkey egg-white lysozyme (TEWL) con-
* Corresponding [email protected].
author.
Fax:
+33
76885122;
E-mail:
taminated by either hen egg-white lysozyme (HEWL) or ribonuclease A [4,5]. HEWL and TEWL are homologous proteins. Only seven out of their 129 residues are different and their three-dimensional structures are very similar (Protein Data Bank entries 61yz and 21z2). Ribonuclease A is a different protein with no structural resemblance to lysozyme, though it has similar molecular weight and isoelectric point. HEWL contamination of TEWL solutions changed the aspect ratio of the resulting crystals towards lower length-to-width ratios (see Fig. 1). The cell parameters and the symmetry of the crystals grown in contaminated solution were the same as those of uncontaminated TEWL crystals (see Section 2). Yet significant amounts of the HEWL contaminant seem to be included in these crystals, approximately proportional to the initial contaminant concentration of the growth solution (see also Ref. [5]). Contamination of TEWL by ribonuclease A, on the other hand,
0022-0248/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S 0 0 2 2 - 0 2 4 8 ( 9 6 ) 0 0 6 8 8 - 4
J. Hirschler, J.C. Fontecilla-Camps / Journal of Co,stal Growth 171 (1997) 559-565
560
{I 12} fsee
{I I0} face
Fig. 1. Schematic drawing of a T E W L crystal (space group P6122). The crystal rests on a {110} face in the growth cell. Co-crystallization of H E W L leads to crystals which are shortened along the c-axis.
did not modify crystal morphology. This shows the importance of the contaminant's structural resemblance to the principal compound in order to interact in the crystal growth process. We proposed that the contaminant's influence is face-specific and that its effect can be compared to what is known from crystal growth of small organic molecules in the presence of tailor-made additives [6]. According to this model, additives with a structure similar to that of the main compound may act on only one set of crystal faces. We speculated that HEWL interacts preferentially with the apical {112} faces of the TEWL crystal, but only to a lesser degree (or not at all) with the lateral {110} faces. This specific interaction would significantly slow down the crystal growth rates of the {112} faces, but not those of the {110} faces. In order to verify our hypothesis, we have carried out a series of face growth rate measurements on TEWL crystal in both uncontaminated solution and in solution contaminated by HEWL. To our knowledge this is the first report on protein crystal growth rates in the presence of homologous contaminants.
tion window and the upper plate are then tightened to the center plate to close the cell and assure air-tightness. Crystal growth is monitored via the two observation windows. With our growth cell design, the protein growth solution cannot be exchanged during the course of an experiment. Consequently, the growing crystal will decrease the overall protein concentration, which, in turn, will reduce the crystal growth rates. We arbitrarily decided that this growth rate reduction should not exceed 7%. Using Eq. (1) with a supersaturation O" = ( C T E w L -- C s o l ) / C s o I = 8.38 (CTEwL i s t h e TEWL concentration, C~o1 is the TEWL solubility of 1.6 m g / m l , see below for exact conditions) corresponding to 15 m g / m l TEWL and a value for r of 2 (see Section 3) shows that a supersaturation decrease of approximately 3% will decrease the growth rate by no more than 6.6%. The calculation of the minimum growth cell volume needed in order not to surpass this 3% decrease is based on the previously published TEWL crystallization conditions by the vapor-diffusion method [4]. A typical crystal obtained in a drop of 4 ILl volume at 15 m g / m l initial TEWL concentration stops growing at a final TEWL concentration of 1.6 m g / m l (see below). This decrease indicates that about 0.054 mg TEWL have been incorporated into the crystal. As this crystal is much larger ( ~ 0.7 mm longest dimension) than the crystals used for our growth rate measurements (see below), this corresponds to the maximum foreseeable TEWL loss in the growth chamber. This chamber has a diameter of 8.5 mm and a height of 4 mm (see 8,5 mm i
.
.
.
i
.
. . . . . . . . . . . .
2. Experimental procedure The crystal growth cells we used are based on a design of Pusey ([7], see Fig. 2). The lower cell plate is first assembled along with the lower observation window and the center plate. This gives a water-tight cavity, into which the protein growth solution is poured. A previously obtained seed crystal is then transferred to this growth solution, where it rests on the lower observation window. The upper observa-
4 mm -"" " [ ' ~ . ~
[ : ~ 1
upper cover slip center plate lower cover slip
Fig. 2. Explosion drawing of the growth cell. Upper, center and lower plates are made of acrylic plastic. The observation windows are the cover slips used on Linbro Plates. The growth chamber is formed by the hole in the center plate and by the two observation windows. The tightening screws have been omitted in the figure for simplicity.
561
J. Hirwhler, J.C. Fontecilla-Camps / Journal of Crystal Growth 171 ( 19971 559-565
Fig. 2), which corresponds to a volume of 227 Ixl. When completely filled with a 15 m g / m l TEWL growth solution, this chamber contains 3.0 mg of available TEWL. The maximum loss of about 0.054 mg TEWL by the crystal growth will thus decrease the initial TEWL concentration by only 1.8%. Even at 10 m g / m l TEWL, this decrease would only amount to 2.8%, which is within the set limits. The growth chamber is also large enough to avoid obstructing convective flow around the growing crystal. This problem may be encountered in a more constrained space, which may reduce transport kinetics
[8]. The TEWL seed crystals were obtained by the vapor-diffusion method at 20°C in sitting drops consisting of a mixture of 2 b~l of 30 m g / m l commercial-grade TEWL (Sigma, crystallized, dialyzed and lyophilized) and of 2 ILl precipitant solution on flat insets [9] in Linbro Plates [10]. The precipitant solution at pH 5.8 consisted of 2.40M NaC1, a 0.1M HEPES buffer (N-[2-hydroxyethyl]piperazine-N'-[2ethanesulfonic acid], Fluka BioChimica) and 15raM NaN 3 used as an antibacterial agent. A reservoir volume of 1 ml of the precipitant solution was used. After equilibration of the drop against the reservoir, (expected final drop volume of 2 b~l, corresponding approximately to initial concentrations of 2.40M NaC1, 0.1M HEPES and 15mM NaN 3 at pH 5.8), the solubility of TEWL crystals was determined to be 1.6 m g / m l [4]. These conditions typically yield 1 to 5 well-shaped crystals of up to 0.7 mm in their longest dimension.
Their cell parameters are a = b = 73.2 ,~ c = 86.8 A, a =/3 = 90.0 ° y = 120.0, space group P6~22 [4]. Occasional appearance of polycrystalline aggregates could not be suppressed either by filtering or dialysis of the protein solution. The seed crystals were slowly conditioned to the growth cell solution by repeated addition of small aliquots of this solution to the drop containing the seed crystal (three times during several hours for TEWL concentrations of 15 mg/ml). This pre-equilibration was needed to minimize the shock of transferring the crystals from the vapor-diffusion drop to the growth cell experiment (marked as " A " in Table 1). Crystals that had grown too large (larger than 300 ~m in their longest dimension) were partially dissolved by diluting the corresponding reservoir solutions ( " B " in Table 1). Under these conditions, the crystals shrank to sizes below 250 ~m over a few days. The rough surfaces they had acquired during this process became smooth again soon after they were transferred to the growth solution. Crystals which were directly transferred to the growth cell without pre-equilibration were also tested ( " C " in Table 11. The cell growth solution consisted of a one-to-one mixture of the precipitant solution described above and of TEWL, with or without 33% HEWL contaminant ( w / w of total protein), dissolved in water. HEWL was added so that the TEWL concentrations stayed constant at their respective levels of 10, 15 or 20 m g / m l (corresponding to o- = 5.25, 8.38 or 11.5, respectively, not taking into account the possible influence of HEWL on TEWL solubility). TEWL
Table 1 TEWL crystal growth rates G for the {112} and {110} faces at 15 m g / m l TEWL in uncontaminated solution Crystal parameters
Growth rates G and correlation coefficients R 2
Initial length (txm)
Initial width (ixm)
Pretreatment
Experiment duration (h)
G{ 112} (tzm/h)
R 2 of G{112}
G{ 110} (Ixm/h)
R 2 of
73 93 133 213 240 253
40 60 73 89 107 80
A A B C B B
53 70 34 22 45 69
4.54 5.41 5.65 3.92 4.83 5.45
1.000 0.999 0.999 0.995 0.999 0.995
0.201 0.287 0.208 0.303 (I.340 (I.275
0.944 0.964 0.812 0.934 0.985 0.904
G{110}
The effect of different initial sizes and pre-treatments on growth rate measurements was verified. Neither the linearity of the individual face growth data over time (R 2) nor the obtained growth rates G showed any systematic variation. The data are plotted in Fig. 3. A: seed crystal adapted to growth cell conditions before its transfer. B: seed crystal larger than 300 ixm in length partially dissolved, and then transferred. C: seed crystal transferred to the growth cell without adaptation.
J. Hirschler, J. C. Fontecilla-Camps / Journal of Crystal Growth 171 (1997)559-565
562
i
400
was used as purchased from Sigma, and HEWL was filtered three times over 3 kDa filter membranes (Microsep 3K for 8 h at 6000g). The growth cells were stored in insulating vessels and kept at 20 _+ 0.5°C. At regular intervals they were removed from the vessels and the length and width of the crystals were measured under a dissecting microscope. An experiment was stopped when extensive secondary nucleation was detected, as this would decrease the protein concentration beyond the set limits. Crystal dimensions were measured from edge to edge and then converted to obtain the orthogonal dimensions from crystal face to crystal face. Known interfacial angles of 60 ° between adjacent {110} faces and of 124 ° between {110} and {112} faces were used for this conversion [4]. These measurements were plotted against time, and the data were approximated to a straight line by the least squares method. The data quality was assessed by the correlation coefficient R 2 of this straight line, whose slope corresponds to the face growth rate G. A first series of experiments was carried out to assess the possible influence of different initial crystal sizes on the growth rates [11] a n d / o r the possible effects due to different pre-treatmerits of the seed crystals.
" • 350 t: o L
250
~
-
i
i
L
i
,~
15 mg/ml T E W L
+ +
uncontarni rmted • {112}
4:
o {110) HEWL-contamin. + {112} x {110}
~A
•
+ +
•A
•
200 150
~.~
tt I00
50
++
~A +
~ ~4:'~i• + .
20
40
60
time
80
1 O0
120
[hours]
Fig. 3. T E W L crystal face growth over time in uncontaminated and in HEWL-contaminated T E W L solution at 15 m g / m l TEWL. The data are summarized in Table 2. In HEWL-contaminated solutions, the {I 12} face growth rates are slowed down, whereas the {110} face growth rates are not significantly changed.
and somewhat lower for data of the lateral faces {110} (abbreviated as G{110}, 0.812 < R 2 < 0.985). Fig. 1 shows the crystal faces and their indices. The observed growth rates lie all within the same range for a given set of faces. This agreement is better for G{112} than for G{110}. This can be seen by calculating the ratio of the root mean square deviations (rmsd) of G to the respective value of G: G{112} is 4.97 t x m / h with a rmsd of 0.662 (0.662/4.97 = 0.133), whereas G{110} is 0.269 ~ m / h with a rmsd of 0.0546 (0.0546/0.269 = 0.202, see Table 2). Neither different initial sizes nor different pre-treatments
3. Results and discussion The validation experiments of the growth cell in contaminant-free 15 m g / m l TEWL solution show that linear face growth rate data can be obtained with high correlation coefficients R 2 (Table 1 and Fig. 3). R 2 is high for growth rate data of the apical faces {112} (abbreviated as G{112}, 0.995 < R 2 < 1 . 0 0 0 ) , Table 2
T E W L crystal growth rates (G) of the apical {112} laces and the lateral {110} faces in uncontaminated and HEWL-contaminated solutions (33% H E W L ( w / w total protein)); root mean square deviations (rmsd) are calculated from repeated experiments
Uncontaminated
Contaminated
CTEWL (mg/ml)
G{112} (~zm/h)
rmsd of G{112}
G{I t0} (I-~m/h)
rmsd of G{110}
Supersaturation cr
G{112}/ G{110}
10 15 20 10 15
0.588 4.97 18.5 0.486 3.02
0.0479 0.662 1.65 0.0438 0.213
-
0.0546 0.672 0.0798
5.25 8.38 11.5 5.25 b 8.38 b
18.5 12.2 _ 10.8
'~ 0.269 1.52 - ~ 0.279
" Growth rates were too low to be determined. b Supersaturation ~r of TEWL, without taking into account a possible influence of H E W L on T E W L solubility.
J. Hirschler, J.C. Fontecilla-Camps / Journal of Co'stal Growth 171 (1997) 559-565
of the seed crystals had any systematic effect o n R 2 of the individual measurements or on the averaged growth rates G. The data presented here show that a structurally related protein contaminant can selectively modify the face growth kinetics of a protein crystal. Contamination of a 15 m g / m l TEWL solution by 7.5 m g / m l HEWL did not significantly modify the face growth rates of the lateral {110} faces of the TEWL crystal (0.279 compared to 0.269 I~m/h in uncontaminated solution), whereas it did reduce those of the apical {112} faces by 39% (3.02 compared to 4.97 ~ m / h , Table 2). R 2 is comparable with those obtained from measurements in uncontaminated solutions (not shown). The ratios of rmsd(G) to G are higher for G{II0} than for G{112}, as already observed in uncontaminated solutions. This specific influence of crystal face growth kinetics shows that the morphological changes previously observed in HEWL-contaminated crystallization of TEWL crystals [4,5,12] are indeed kinetic phenomena. Briefly, in our model we attribute these changes to the structural relationship between TEWL and HEWL, which may exert different interactions
L
~
i
20 mglmlTEWL
90
• ( I 12}, uncontaminated TEWL o { 1 10}, uncontarninated TEWL
8o
e.
70
0 I,.
60
(j
50
t
- =°1 ;o'd-;.,; mO J ~J ~4001 TEWL
At 4O 30
20-
"
:°o°ot
e 2
0
L SO
I00
1
10
4- (I 12) HEWL-oontarnin. [
I~
200
2~
300
3~
time [hours]
AA
o ( oi~I~.
oo8O -
,
4
.
,
8
,
,
.
~
•
,
12
16
20
time
[hours]
,
,
24
2'8
Fig. 4. Face growth data observed with 20 m g / m l uncontaminated TEWL and 10 m g / m l contaminated TEWL solutions (inset). The data show the strong dependence on the TEWL concentration. At 10 m g / m l TEWL, the contaminant reduces the {112} growth rates less than it does at 15 m g / m l (see Fig. 3). {110} growth rates at 10 m g / m l TEWL could not be measured.
563
with the various faces of the growing TEWL crystal [4]. The {112} faces display more affinity for the contaminant than the {110} faces. Thus the {112} faces are less accessible to TEWL molecules due to their higher partial contaminant coverage. Their growth will then be slower than that of the {110} faces, as we have shown with our measurements. This represents, according to our knowledge, the first report on face growth rates of protein crystals in a contaminated solution. Five m g / m l HEWL contamination of a 10 m g / m l TEWL solution reduces the growth rates of the {112} faces by 17% (from 0.588 to 0.486 tzm/h, Table 2 and Fig. 4). {110} face growth rates were too low to be reliably measured (see below). Consequently, we do not know the contaminant's effects on the {110} faces at this TEWL concentration. Yet from the results at 15 m g / m l TEWL we have seen that the {110} face growth rates were not significantly influenced by the contaminant. By extension, we can assume equally unmodified {110} face growth rates at 10 m g / m l TEWL. Thus the G{112} decrease of 17% at 10 m g / m l TEWL compares to the 39% decrease at 15 m g / m l TEWL. This indicates an increasing effect of the contaminant with increasing supersaturation. If confirmed by further measurements, this supersaturation dependence may be opposed to that of crystal growth of contaminated low molecular weight materials. In fact, according to a recent model by Kubota and Mullin, the lower the supersaturation of the principal compound, the more pronounced the effect of the contaminant is [13]. Yet the comparability between the two domains of crystal growth is limited. Firstly, the supersaturation range employed in protein crystal growth is orders of magnitude higher than that found in the crystal growth of low molecular weight materials. Secondly, the respective crystallizing species may be differently aggregated: they are assumed to be monomers in the case of low molecular weight materials, whereas proteins may exist as larger aggregates. The aggregates' sizes may furthermore change with supersaturation [8,14,15]. The uncontaminated TEWL growth rates observed here at 15 mg/ml TEWL are of the same order of magnitude as those published for HEWL [16]. However, in our case the variation of the growth rates as a function of supersaturation seems
564
J. Hirschler, J.C. Fontecilla-Camps / Journal of Crystal Growth 171 (1997) 559-565
to be different (Table 2 and below). The empirical equation
G = k o r,
(l)
is commonly used to fit the dependence of the growth rate on the supersaturation (growth rate G, supersaturation o', constant k). The slope of a log(o-)-log(G) plot of TEWL growth rates gives for r a value of 4.4 (R 2 = 0.999). The corresponding value for HEWL growth rates is r = 2.0 [17]. The value of r = 2 is generally taken to indicate a spiral growth mechanism, whereas r > 2 indicates growth via surface nucleation [18]. Thus HEWL and TEWL crystallization under the respective conditions may proceed via different growth mechanisms. However, this approach can only serve as a first indicator of the growth mechanism, as only techniques such as atomic force microscopy or interferometry can yield information on a molecular level. With a factor r of 4.4, we also have to modify our initial assumption for the cell volume calculation based on r = 2 (see Section 2). It actually indicates a maximum error in G of 12% instead of the previous 6.6%. However, as our assumption takes into account a far larger crystal than those crystals measured here, the actual deviation in G will have been smaller than 12%. The ratio G{ 112}/G{ 110} in uncontaminated solution is 18.5 and 12.2 at 15 and 20 m g / m l TEWL, respectively (Table 2). At 10 m g / m l TEWL, G{II2} could be reliably measured for up to 340 h, whereas the G{ll0} were too low to be measured. These ratios show that the different supersaturation levels influence the two face growth rates (G{II2} and G{110}) differently, as it was also observed on tetragonal HEWL crystals [19]. This effect may be explained by different absorption of intrinsic impurities on the crystal faces [20]. According to a recent report, crystal-impurity interactions can also depend on the crystal size [3]: crystals with maximum lengths of under 40 ~m were shown to include higher levels of NaC1 and inherent protein impurities than larger crystals, possibly infringing crystal growth. Yet this does not pertain our experiments, as our smallest crystal was twice as long. We altered the TEWL supersaturation by varying the TEWL concentration in our experiments. Another possibility is to vary protein solubility using
temperature (see Ref. [16] for measurements of HEWL growth rates). We chose not to use this method, since the kinetics of every other temperature-dependent process during crystallization may also be modified [20].
4. Conclusion This first determination of crystal growth rates in contaminated protein solution confirms that structurally related protein contaminants influence growth rates of protein crystals in a face-specific manner. Co-crystallization of the contaminant HEWL with TEWL leads to a significant drop of growth rates of the apical {112} faces of the crystal, but does not influence the growth rates of the lateral {110} faces. These results on contaminated protein crystal growth may be explained by what is known concerning the face-specific influence of structurally related contaminants in organic crystal growth. Together with more detailed knowledge on molecular-level protein contaminant-protein crystal interactions, crystal engineering may thus become a feasible means to modify protein crystal morphology systematically for research or for industrial applications. Further measurements should validate our preliminary results concerning the contaminant's supersaturation dependence. If so, it may be advantageous to crystallize proteins in a supersaturation range where the deleterious effects of contaminants, inherent to protein crystallization, are minimized.
Acknowledgements We thank Dr. Pusey for providing help on the initial design of our growth cell and Dr. Sunawaga for pointing out that "aspect ratio" is the appropriate expression to use for evaluating crystal morphology.
References [1] R. Gieg6, B. Lorber and A. Theobald-Dietrich, Acta Cryst. D 50 (1994) 339. [2] P.G. Vekilov, M. Ataka and T. Katsura, Acta Cryst. D 51 (1995) 207.
J. Hirschler, J.C. Fontecilla-Camps / Journal of Cr3'stal Growth 171 (1997)559-565 [3] P.G. Vekilov, L.A. Monaco, B.R. Thomas, V. Stojanoff and F. Rosenberger, Acta Cryst. D, submitted. [4] J. Hirschler and J.C. Fontecilla-Camps, Acta Cryst. D 52 (1996) 806. [5] C. Abergel, M.P. Nesa and J.C. Fontecilla-Camps, J. Crystal Growth 110 (1991) 11. [6] M. Lahav and L. Leiserowitz, J. Phys. D 26 (1993) B22. [7] M.L. Pusey, Anal. Biochem. 158 (1986) 50. [8] S. Miyashita, H. Komatsu, Y. Suzuki and T. Nakada, J. Crystal Growth 141 (1994) 419. [9] T.M.B. Soriano and J.C. Fontecilla-Camps, J. Appl. Cryst. 26 (1993) 558. [10] J. Hirschler, M.H. Charon and J.C. Fontecilla-Camps, Prot. Sci. 4 (1995) 2573. [1 I] M.L. Pusey and R. Naumann, J. Crystal Growth 76 (1986) 593.
565
[12] K. Provost, M.C. Robert, J. Crystal Growth 156 (1995) 112. [13] N. Kubota and J.W. Mullin, J. Crystal Growth 152 (1995) 203. [14] M. Li, A. Nadarajah and M.L. Pusey, J. Crystal Growth 156 (1995) 121. [15] F. Rosenberger, P.G. Vekilov, M. Muschol and B.R. Thomas, J. Crystal Growth 168 (1996) I. [16] E. Forsythe and M.L. Pusey, J. Crystal Growth 139 (1994) 89. [17] M.L. Pusey, R.S. Snyder and R. Naumann, J. Biol. Chem. 261 (1986) 6524. [18] A.E. Nielsen, J. Crystal Growth 67 (1984) 289. [19] S.D. Durbin and G. Feher, J. Crystal Growth 76 (1986) 583. [20] L.A. Monaco and F. Rosenberger, J. Crystal Growth 129 (1993) 465.