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Precrystallization of human apoprotein A-I based on its aggregation behavior in solution studied by dynamic light scattering
Journal of
MOLECULAR STRUCTURE ELSEVIER
Journal of Molecular Structure 440 (1998) 1-8
Precrystallization of human apoprotein A-I based on its aggregation behavior in solution studied by dynamic light scattering Vfctor M. Bolafios-Garcfa a'b, Jaime Mas-Oliva b, Manuel Soriano-Garcfa a, Abel Moreno a'* "Depto. de Bioestructura, lnstituto de Qulmica, Universidad Nacional Aut6noma de M~xico, Apdo. Postal 70-243, C.P. 04510, Ciudad Universitaria, Deleg., Coyoacdn, MFxico ~Depto. de Biquimica, lnstituto de Fisiologia Celular, Universidad Nacional Aut6noma de MFxico, Apdo. Postal 70-243, C.P. 04510, Ciudad Universitaria, Deleg., Coyoac6n, M~xico
Received 11 December 1996; revised 31 March 1997; accepted 31 March 1997
Abstract
This work reports several aspects concerning dynamic light scattering data of apoprotein A-I in solution. Using the geometrical factor (H) and turbidity (r) at different pH values ranging from 5 to 10, we have tried to elucidate either if the initial formation of clusters and the trend for aggregation are carried out by nucleation or random mechanisms. Taking into account the solubility of the protein, this behavior depends on the precipitating agent used and the rate of mass transporting capacity carried out by vapor diffusion in small drops. Finally, we demonstrate that dynamic light scattering is a useful tool in order to determine protein precrystallization conditions. © 1998 Elsevier Science B.V. Keywords: DLS techniques; Apoprotein A-I; Protein crystallization
1. Introduction
Protein crystallization has become a very useful and powerful tool in the determination of threedimensional protein structures based on the build-up of supra-atomic and supramolecular ordered states. Protein structures obtained from crystal X-ray analysis has also become essential in areas like protein engineering, based-structure drug design and prot e i n - l i g a n d or p r o t e i n - p r o t e i n molecular recognition. Although the first protein was crystallized over a hundred years ago, crystallization has largely remained an
* Corresponding author: Tel.: (52-5) 6 22 44 03; fax: (52-5) 6 16 22 03; e-mail: [email protected]
art rather t h a n become a science mainly due to the complex multicomponent nature of a crystallizing system and the long time required to observe changes in different parameters by visual inspection. Nowadays the process of crystallization still represents the bottleneck in the macromolecular structural analysis carried out by X-ray diffraction techniques [1]. Ordinarily, it is still necessary to perform wide screenings in order to find the best crystallization conditions for macromolecules [2]. Evidently, this time demanding process requires large amounts of protein. Nevertheless, based on empirical observations, it has been suggested that macromolecules that are monodisperse crystallize readily, whereas polydisperse systems rarely do [3]. This hypothesis has been useful in several cases for different groups [4-7]. Of course, this observation
0022-2860/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved PH S0022-2860(97)00246-9
2
V.M. Bola~os-Garc[aet al./Journal of Molecular Structure 440 (1998) 1-8
needs to be taken carefully for evaluating new alternative ways of crystallization. Dynamic light scattering (DLS, also called photon correlation spectroscopy) is a technique used to measure the translational diffusion coefficient (DT) of a macromolecule undergoing Brownian motion in solution. Monochromatic light scattered by moving protein molecules displays intensity fluctuations according to their motion. Then, an analysis of the light scattering signal provides quantitative information about the behavior in solution of the dissolved protein. In addition, if the intensity of the scattered light depends only on the spatial arrangement of the atoms, the phenomena is referred as Rayleigh-Gans-Debye scattering [8]. As additional advantages, a typical DLS experiment takes just a few minutes, is a non-destructive procedure and requires a minimum of pure sample. We are interested in the structure-function relationship of human apolipoproteins, considered the protein components of plasma lipoproteins which allow the transport of water insoluble lipids in the blood plasma. Apoprotein A-I (ApoAI), which is the major protein of high density lipoproteins (HDL) [9], contains 243 aminoacid residues in length and presents a molecular weight of 28 KDa [10]. In addition to its role in lipid transfer between lipoproteins, it has other relevant biological activities as in the activation of lecithin-cholesteryl-acyltransferase enzyme (LCAT) [11] and in the size distribution of HDL particles [12]. Since human apolipoprotein A-I has not yet been crystallized, the present study describes how dynamic light scattering experiments can be useful to determine the precrystallization conditions under which crystal nuclei can be obtained.
Instrument (Protein Solutions, Co.). Samples were injected through Whatman Anotop 10 plus 20 nm filters. Multiple measurements were taken from different samples. Data were collected and analyzed using the AutoPro data software for the DynaPro 801 instrument (Protein Solutions Co.). Refractive index were measured using an Abbey's refractometer (Carl Zeiss) calibrated with monobromonaphthalin (rid = 1.660). 2.3. C~stal growth techniques
Linbro 24 well plates (Hampton Res. Co.) for both hanging-drop and sitting-drop methods were used. Samples of apoAI (4/zl) were used and an equal volume of precipitating agent was added to the drop in every experiment. The different reservoirs were filled with l ml of precipitating agent. For the hanging-drop technique, siliconized 22 mm circle cover slides were employed and sealed to the reservoir with vacuum grease. With respect to the sitting-drop method, polystyrene micro-bridges (Hampton Res. Co.) were placed in the Linbro plates and sealed with plain cover slides and vacuum grease. All the experiments were done at 25°C, leaving the samples non-disturbed for at least two days. 2.4. Bioassays
Protein concentrations were estimated by both Lowry [13] and Bradford [14] methods using Protein Assay commercial kits (Bio-Rad Lab).
3. Results and discussion 2. Experimental 3.1. Hydrodynamic properties as a function of pH 2.1. Materials used for analysis
Lyophilized apoAI was obtained from PerImmune, Inc. (Rockville, MD), lithium sulfate and all other salts from Sigma Chemical Co. (St. Louis, MO). 2.2. Instrumentation
Dynamic light scattering measurements were performed using a DynaPro-801 Dynamic Light Scattering
A previously reported study of apoAI, using quasielastic light scattering evaluated self-aggregation and its effects on plasma transport of bile salts [15]. These results have disagreed with other authors [ 16,17] and the reported conditions in our hands failed to yield nuclei crystals, using both the hanging- and sittingdrop methods. Due to these unsuccessful trials, the hydrodynamic radius, diffusion coefficient, estimated molecular weight and polydispersity at different pH
V.M. Bolalios-Garcia et al./Journal of Molecular Structure 440 (1998) 1-8
values have been evaluated under the conditions mentioned next. A quick protein aggregation is observed when the pH is closer to the isoelectric point of apoAI (pl = 5.6), in agreement with the protein solubility theory (reviewed by Arakawa and Timasheff, [18]). As observed in Fig. 1A, when the pH decreases, the diffusion coefficient also decreases due to protein aggregation. At higher pH values an increase indicates that apoAI molecules in solution are diffusing better if they have smaller sizes. Since a few years ago it was suspected that laser light scattering produces heating and multiple scattering effects on protein samples, it is important to note that further studies have demonstrated a negligible effect of these factors on diffusion coefficient measurements [19]. An opposite tendency in the diffusion coefficient is observed for the hydrodynamic radius, where this value decreases as soon as the pH increases (Fig. 1B). An estimation of the apoAI aggregates size is given from the estimated molecular weight (Fig. 1C) and the size distribution obtained from polydispersity measurements (Fig. 1D). These parameters indicate that apoAI self-aggregation is taking place at pH values closer to the isoelectric point where its solubility is the lowest. Moreover, a wider size distribution, measured as polydispersity, is also observed for the aggregates at lower pH values (pH 5), which is considered a condition that might be undesirable for apoAI crystal nuclei formation. From this, it is clear that pH values where apoAI is more soluble (pH values far from its isoelectric point) must be chosen since more molecules will be in solution and available to form a crystalline phase. However, pH values where the protein is freely soluble must be avoided due to a supersaturation state, that if occurs, it is reached after a long time. 3.2. Determination of type of aggregation
Based on DLS experiments, some physicochemical parameters, such as the geometrical factor, H, and turbidity, r, were estimated. These parameters gave helpful information about apoA1 aggregation and provided important clues to define the conditions where crystal nuclei would be obtained. For aqueous solutions of proteins, it is permissible to regard the particles as sufficiently small so that Eq. (1) is applicable to visible light scattering systems of spherical
3
particles possessing molecular weights up to about five million. Independence of the particles is insured if the light scattering data is extrapolated to idealized conditions (infinite dilution). Under these conditions, the geometrical factor H can be calculated from the following expression: 3 2
H = 327r r/007 - rl0) 3NA Xac 2
2
( 1)
where r/0 = refractive index of medium, rl = refractive index of protein solution, ~, = wavelength, N A = Avogadro's number and c = particle concentration (in weight). In the case of the DynaPro-801 instrument, both the wavelength and the scattering angle are constant (780 nm and 90 °, respectively). For apoAI at 2 mg m1-1 in Trizma-HC1 5 0 m M pH = 8.0, the estimated refractive index is rl = 1.360 and the buffer alone rl0 = 1.335. In this way, the calculated geometrical factor H value is 4.80 x 1 0 -9 tool g-2 cm 2 molecule-~. Once calculated H and taking account that for small independent particles which are both optically isotropic and sufficiently small compared with the wavelength of the incident light, application of Rayleigh's theory gives: "r= HcM
(2)
where turbidity is represented as 7-, c is the particle concentration (in weight) and M is the molecular weight of protein aggregates. For a system in which the particles are aggregating in a random fashion, i.e. any size particle or aggregate combining with any other size particle or aggregate, the turbidity will increase linearly with time. Similarly, if the particles are aggregating by nucleation (in an ordered way), the turbidity will increase quadratically with time. According to Fig. 2, r changes follow an asympthothic line when Li2SO4 as a precipitant agent is used. This behavior indicates that apoAI aggregation is occurring in an ordered way and nucleation is taking place. In this way, physical information (hydrodynamic behavior) may be successfully linked with chemical information (crystal nuclei formation). 3.3. The salting-in and salting-out regions
According to the Debye-Hi.ickel theory, protein solubility has a minimum at the isoelectric point at low ionic strength, which is usually observed [17]. At
4
V.M. Bola~os-Garcia et al./Journal of Molecular Structure 440 (1998) 1-8
60
b
18
X
=, so
~_ 16
°~5,~'- 40
~
14
~
12
~ 3o
i:5
10 5
6
7
8
9
10
5
6
7
8
pH
9
10
pH
33
45
d
30 .~
E c-
39
t_
27 2,4 21
@
18
33 1D
nO
15 12 9
24
6 5
6
7
8
pH
9
10
•
5
|
6
,
l
7
.
,
8
~
I
9
|
|
10
pH
Fig. 1. ApoAI protein solution properties measured by dynamic light scattering at different pH values: (a) diffusion coefficient, (b) hydrodynamic radius, (c) estimated molecular weight, and (d) polydispersity.
V.M. Bola~os-Garcia et al./Journal of Molecular Structure 440 (1998) 1-8
70 6O o
50
X
6
40
~
30
-e
20
i--
11)
0
-
0
0,1
0,2
0,3 0,4
0,5
U 2SO4 (moles/L.) Fig. 2. Turbidity of apoAl solution against Li2SO4 as the precipitanting agent. The tendency indicate that protein aggregation is occurring in an ordered way and nucleation events may be taking place.
low salt concentration all salts have a salting-in effect on proteins that result in a solubility increase. On the other hand, at high salt concentrations protein solubility is known to decrease and this effect is called salting-out. As shown in Fig. 3A-D, it is possible to evaluate both the salting-in and the salting-out regions for apoAI using Li2SO4 as a precipitating agent. According to the Fig. 3A, the diffusion coefficient increases with each Li2SO4 addition until its concentration reaches 0.37 M. This behavior is due to a salting-in effect, where protein solubility increases, as mentioned before. After this Li2SO4 concentration is reached, the diffusion coefficient starts to decrease since apoAI aggregation starts to take place. This change corresponds to the entry in the salting-out region. These results are in agreement with our hydrodynamic radius values. When Li2SO+ reaches 0.37 M, apoAI starts to aggregate and the hydrodynamic radius fastly increases from 12 × 10 -~8 cm to 145 × 10 -~8 cm (Fig. 3B). In the case of the estimated molecular weight, as shown in Fig. 3C, also changes from about 28 kDa to around 500 kDa. Finally, polydispersity also increases around 10 times in the
5
precipitant agent concentration range mentioned before (Fig. 3D). All of these parameters taken together, bring information related to the specific protein aggregation rates. When this aggregation occurs in a well ordered way, crystal nuclei formation might take place. These observations are related with metastable zone in Oswald-Miers phase diagrams, where nucleation phenomena takes place [20]. Once a nucleus reaches a specific size, going through the critical size, it grows spontaneously to form a macroscopic solid phase. If the rate of aggregation is stepwise controlled and diffusion maintained, the cluster will form a crystal; if not, a quick growth rate of clustering will be achieved and an amorphous precipitate will be obtained. It is well known that an optimal amount of protein is desirable for growing large enough crystals for X-ray analysis. In the case of the experimental conditions employed for dynamic light scattering experiments, the optimal protein concentration to be used depends on the molecular weight of the sample. For instance, from 0.1 for large macromolecules up to 4 mg ml -j for proteins with low molecular weight. In order to crystallize a new protein, using data coming from DLS analysis, it is possible to scale not only the concentration of protein solution but also the precipitating agent concentration to reach a suitable degree of supersaturation to provoke macroscopic crystal growth. As a consequence of this observation, crystallization trials for apoAI were made using two to three times the concentration of both protein and precipitant agent with respect to the concentration used in DLS experiments. At the beginning, several experiments were done using the same concentration employed for DLS measurements in order to initiate our crystallization trials, but extremely small crystals were obtained (Fig. 4A). Therefore, maintaining the ratio protein/precipitant agent new crystallization trials with both the hanging-drop and sitting-drop techniques were performed [21]. After 48 hours with both techniques crystal nuclei were formed when apoA] concentration was maintained between 2 and 4 mg ml i and Li2SO4 kept between 0.5-1.0 M as shown in Fig. 4B. Since these dentritic-shaped crystals obtained by DLS analysis apparently are grown by a two-dimensional mechanism, the demonstration of this assumption will be only obtained using atomic force microscopy [22].
6
V.M. Bola~os-Garcia et al./Journal of Molecular Structure 440 (1998) 1-8
140
x
160 =.-. -o x E
120 100
~
.~
80
140 120
lOO
80
60
~
60
20
I
20
0
0,1
0,2
0,3
0,4
0,5
0
0,1
Li2SO 4 (moles/L)
0,2
0,3
0,4
0,5
Li 2S04 (moles/L)
9O "
5oo
c
d
8O
-| i
E ~j
300
===>'
200
~
40
no
30
100
~
0
0
•
20 " "" ' 0,1 0,2
10 0,3
0,4
Li 2S0 4 (moles/L)
0,5
0
. . . . . . ° ' ' 0,1 0,2 0,3 0,4
' 0,5
Li 2S0 4 (moles/L)
Fig. 3. ApoAI solution properties at increased Li2SO4 concentration: (a) diffusion coefficient, (b) hydrodynamic radius, (c) estimated molecular weight, and (d) polydispersity. Both salting-in and salting-out regions can be appreciated (see the text for details).
V.M. Bolaaos-Garcla et al./Journal of Molecular Structure 440 (1998) 1-8
a
7
!ilil
Fig. 4, Photograph of apoAl: (A) small crystals obtained by direct concentrations from DLS in precrystallization experiments, and (B) scaling two or three times the protein and precipitating concentration, two-dimensional dendritic-shaped crystals were obtained by the hanging-drop technique under the following conditions: protein concentration 4 mg ml-~ in Trizma 50mM pH 8.0, Li 2SO4 1.0 M, temperature 20°C. Similar morphology was observed using the sitting-drop technique.
8
V.M. Bola~os-Garcia et al./Journal of Molecular Structure 440 (1998) 1 - 8
Employing apoAI at 6 mg ml -~ and Li2SO 4 at 1.5 M, an amorphous precipitate is obtained, that might be due to the fact that an equilibrium state is quickly reached. Using the hanging-drop technique, the crystal nuclei formed are slightly bigger than using the sitting-drop technique. Although these nuclei are not large enough for X-ray crystallographic analysis, protein nucleus formation can be considered as a precrystallization step. Our next goal will be focused on growing these nuclei until they can reach a suitable size for X-ray diffraction analysis. This will be done by the crystallization method in capillary tubes as has been showed by Garcfa-Ruiz e t al. [23]. It is important to emphasize that the slow supply of molecules by diffusion causes a reduction of the protein concentration in the vicinity of a rapidly growing crystal. In this way, the reduced protein concentration prevents the formation of new nuclei and therefore, eliminates unwanted interference between crystals. Besides, crystals that grow too fast usually reach a smaller terminal size, presumably due to structural defects that can be incorporated. Consequently, the size of the crystal depends on the degree of supersaturation as well as on the kinetic pathway of nucleation and growth. In this fashion it is very important to determine the effect of several parameters such as pH, ionic strength, temperature and precipitating agent concentration on protein solubility in order to perform a rational screening for crystallization. As demonstrated here, the use of DLS can be considered a very useful technique to reach this goal. Using this technique it is possible to match physical measurements of macromolecules with their chemical properties in order to yield crystals.
Acknowledgements This study has been partially supported by D G A P A - U N A M (Grant IN-201294). Vfctor Bolafios was partially supported by PADEP, UNAM (no. 030381). Abel Moreno acknowledges finantial support from C O N A C y T in Programa de Repatriaci6n
de Cientfficos Mexicanos. This is a contribution (1597) from Instituto de Qufmica, UNAM.
References [1] A. McPherson, A.J. Malkin, Y.G. Kuznetsov, Structure 3 (1995) 759. [2] J. Jancarik, S.-H. Kim,Journalof Applied Crystallography.24 (1991) 409. [3] A. Ferr6-D'Amar~,S.K. Burley, Nature of Structural Biology 2 (1994) 357. [4] W. Kadima, A. McPherson, M.F. Dunn, F.A. Juruak, Biophysics Journal 57 (1990) 125. [5] V. Mikol, E. Hirsch, R. Gieg6, Journal of Molecular Biology 213 (1990) 187. [6] A. Malkin,A. McPherson,Acta CrystallographicaD50 (1994) 385. [7] W.W. Wilson, Methods: A Companion to Methods in Enzymology,Vol. 1, AcademicPress, Orlando, Florida, 1993. [8] J.B. Bishop,J.C. Martin,W.M. Rosenblum,Journal of Crystal Growth 110 (1991) 164. [9] H.N.Baker, T. Delahunty,A.M. Gotto, R.L. Jackson,Proceedings of National Academyof Science, USA 71 (1974) 3631. [10] H.B. Brewer, T. Fairwell,A. LaRue, R. Ronan,A. Houser,T.J. Bronzert, Biochemistry and Biophysics Research Comm. 80 (1978) 623. [11] E. Steyrer, G.M. Kosmer,Biochim. Biophys. Acta 958 (1988) 484. [12] B.L. Thurberg, C.A. Reardon, S. Getz, Journal of Biology and Chemistry 271 (1996) 6062. [13] O.H. Lowry,N.J. Rosebrough,A.L. Farr, R.J. Randall,Journal of Biology and Chemistry 193 (1951) 265. [14] M. Bradford, Annalsof Biochemistry72 (1976) 248. [15] J.M. Donovan,G.B. Benedek, M.C. Carey, Biochemistry 26 (1987) 8116. [16] D. Atkinson, D.M. Small, Annual Review of Biophysics Biophysical Chemistry 15 (1986) 403. [17] A.M. Scanu, C. Edelstein, B.W. Shen, Lipid-Protein Interactions,Wiley, New York, 1982. [18] T. Arakawa, S.N. Timasheff, Methods of Enzymology 114 (1985) 49. [19] Y.S. Oh, S. Johnson, Journal of Chemistry and Physics 74 (1981) 2717. [20] A. Ducruix, R. Gieg& Crystallizationof Nucleic Acids. A Practical Approach, IRL Press, Oxford, 1992. [21] A. McPherson, Preparation and Analysisof Protein Crystals, Wiley,, New York, 1982. [22] H.G. Hansma, J.H. Hoh, Annual Review of Biophysics and BiomolecularStructures 23 (1994) 115. [23] J.M. Garcfa-Ruiz, A. Moreno, Acta Crystallographica D50 (1994) 484.
MOLECULAR STRUCTURE ELSEVIER
Journal of Molecular Structure 440 (1998) 1-8
Precrystallization of human apoprotein A-I based on its aggregation behavior in solution studied by dynamic light scattering Vfctor M. Bolafios-Garcfa a'b, Jaime Mas-Oliva b, Manuel Soriano-Garcfa a, Abel Moreno a'* "Depto. de Bioestructura, lnstituto de Qulmica, Universidad Nacional Aut6noma de M~xico, Apdo. Postal 70-243, C.P. 04510, Ciudad Universitaria, Deleg., Coyoacdn, MFxico ~Depto. de Biquimica, lnstituto de Fisiologia Celular, Universidad Nacional Aut6noma de MFxico, Apdo. Postal 70-243, C.P. 04510, Ciudad Universitaria, Deleg., Coyoac6n, M~xico
Received 11 December 1996; revised 31 March 1997; accepted 31 March 1997
Abstract
This work reports several aspects concerning dynamic light scattering data of apoprotein A-I in solution. Using the geometrical factor (H) and turbidity (r) at different pH values ranging from 5 to 10, we have tried to elucidate either if the initial formation of clusters and the trend for aggregation are carried out by nucleation or random mechanisms. Taking into account the solubility of the protein, this behavior depends on the precipitating agent used and the rate of mass transporting capacity carried out by vapor diffusion in small drops. Finally, we demonstrate that dynamic light scattering is a useful tool in order to determine protein precrystallization conditions. © 1998 Elsevier Science B.V. Keywords: DLS techniques; Apoprotein A-I; Protein crystallization
1. Introduction
Protein crystallization has become a very useful and powerful tool in the determination of threedimensional protein structures based on the build-up of supra-atomic and supramolecular ordered states. Protein structures obtained from crystal X-ray analysis has also become essential in areas like protein engineering, based-structure drug design and prot e i n - l i g a n d or p r o t e i n - p r o t e i n molecular recognition. Although the first protein was crystallized over a hundred years ago, crystallization has largely remained an
* Corresponding author: Tel.: (52-5) 6 22 44 03; fax: (52-5) 6 16 22 03; e-mail: [email protected]
art rather t h a n become a science mainly due to the complex multicomponent nature of a crystallizing system and the long time required to observe changes in different parameters by visual inspection. Nowadays the process of crystallization still represents the bottleneck in the macromolecular structural analysis carried out by X-ray diffraction techniques [1]. Ordinarily, it is still necessary to perform wide screenings in order to find the best crystallization conditions for macromolecules [2]. Evidently, this time demanding process requires large amounts of protein. Nevertheless, based on empirical observations, it has been suggested that macromolecules that are monodisperse crystallize readily, whereas polydisperse systems rarely do [3]. This hypothesis has been useful in several cases for different groups [4-7]. Of course, this observation
0022-2860/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved PH S0022-2860(97)00246-9
2
V.M. Bola~os-Garc[aet al./Journal of Molecular Structure 440 (1998) 1-8
needs to be taken carefully for evaluating new alternative ways of crystallization. Dynamic light scattering (DLS, also called photon correlation spectroscopy) is a technique used to measure the translational diffusion coefficient (DT) of a macromolecule undergoing Brownian motion in solution. Monochromatic light scattered by moving protein molecules displays intensity fluctuations according to their motion. Then, an analysis of the light scattering signal provides quantitative information about the behavior in solution of the dissolved protein. In addition, if the intensity of the scattered light depends only on the spatial arrangement of the atoms, the phenomena is referred as Rayleigh-Gans-Debye scattering [8]. As additional advantages, a typical DLS experiment takes just a few minutes, is a non-destructive procedure and requires a minimum of pure sample. We are interested in the structure-function relationship of human apolipoproteins, considered the protein components of plasma lipoproteins which allow the transport of water insoluble lipids in the blood plasma. Apoprotein A-I (ApoAI), which is the major protein of high density lipoproteins (HDL) [9], contains 243 aminoacid residues in length and presents a molecular weight of 28 KDa [10]. In addition to its role in lipid transfer between lipoproteins, it has other relevant biological activities as in the activation of lecithin-cholesteryl-acyltransferase enzyme (LCAT) [11] and in the size distribution of HDL particles [12]. Since human apolipoprotein A-I has not yet been crystallized, the present study describes how dynamic light scattering experiments can be useful to determine the precrystallization conditions under which crystal nuclei can be obtained.
Instrument (Protein Solutions, Co.). Samples were injected through Whatman Anotop 10 plus 20 nm filters. Multiple measurements were taken from different samples. Data were collected and analyzed using the AutoPro data software for the DynaPro 801 instrument (Protein Solutions Co.). Refractive index were measured using an Abbey's refractometer (Carl Zeiss) calibrated with monobromonaphthalin (rid = 1.660). 2.3. C~stal growth techniques
Linbro 24 well plates (Hampton Res. Co.) for both hanging-drop and sitting-drop methods were used. Samples of apoAI (4/zl) were used and an equal volume of precipitating agent was added to the drop in every experiment. The different reservoirs were filled with l ml of precipitating agent. For the hanging-drop technique, siliconized 22 mm circle cover slides were employed and sealed to the reservoir with vacuum grease. With respect to the sitting-drop method, polystyrene micro-bridges (Hampton Res. Co.) were placed in the Linbro plates and sealed with plain cover slides and vacuum grease. All the experiments were done at 25°C, leaving the samples non-disturbed for at least two days. 2.4. Bioassays
Protein concentrations were estimated by both Lowry [13] and Bradford [14] methods using Protein Assay commercial kits (Bio-Rad Lab).
3. Results and discussion 2. Experimental 3.1. Hydrodynamic properties as a function of pH 2.1. Materials used for analysis
Lyophilized apoAI was obtained from PerImmune, Inc. (Rockville, MD), lithium sulfate and all other salts from Sigma Chemical Co. (St. Louis, MO). 2.2. Instrumentation
Dynamic light scattering measurements were performed using a DynaPro-801 Dynamic Light Scattering
A previously reported study of apoAI, using quasielastic light scattering evaluated self-aggregation and its effects on plasma transport of bile salts [15]. These results have disagreed with other authors [ 16,17] and the reported conditions in our hands failed to yield nuclei crystals, using both the hanging- and sittingdrop methods. Due to these unsuccessful trials, the hydrodynamic radius, diffusion coefficient, estimated molecular weight and polydispersity at different pH
V.M. Bolalios-Garcia et al./Journal of Molecular Structure 440 (1998) 1-8
values have been evaluated under the conditions mentioned next. A quick protein aggregation is observed when the pH is closer to the isoelectric point of apoAI (pl = 5.6), in agreement with the protein solubility theory (reviewed by Arakawa and Timasheff, [18]). As observed in Fig. 1A, when the pH decreases, the diffusion coefficient also decreases due to protein aggregation. At higher pH values an increase indicates that apoAI molecules in solution are diffusing better if they have smaller sizes. Since a few years ago it was suspected that laser light scattering produces heating and multiple scattering effects on protein samples, it is important to note that further studies have demonstrated a negligible effect of these factors on diffusion coefficient measurements [19]. An opposite tendency in the diffusion coefficient is observed for the hydrodynamic radius, where this value decreases as soon as the pH increases (Fig. 1B). An estimation of the apoAI aggregates size is given from the estimated molecular weight (Fig. 1C) and the size distribution obtained from polydispersity measurements (Fig. 1D). These parameters indicate that apoAI self-aggregation is taking place at pH values closer to the isoelectric point where its solubility is the lowest. Moreover, a wider size distribution, measured as polydispersity, is also observed for the aggregates at lower pH values (pH 5), which is considered a condition that might be undesirable for apoAI crystal nuclei formation. From this, it is clear that pH values where apoAI is more soluble (pH values far from its isoelectric point) must be chosen since more molecules will be in solution and available to form a crystalline phase. However, pH values where the protein is freely soluble must be avoided due to a supersaturation state, that if occurs, it is reached after a long time. 3.2. Determination of type of aggregation
Based on DLS experiments, some physicochemical parameters, such as the geometrical factor, H, and turbidity, r, were estimated. These parameters gave helpful information about apoA1 aggregation and provided important clues to define the conditions where crystal nuclei would be obtained. For aqueous solutions of proteins, it is permissible to regard the particles as sufficiently small so that Eq. (1) is applicable to visible light scattering systems of spherical
3
particles possessing molecular weights up to about five million. Independence of the particles is insured if the light scattering data is extrapolated to idealized conditions (infinite dilution). Under these conditions, the geometrical factor H can be calculated from the following expression: 3 2
H = 327r r/007 - rl0) 3NA Xac 2
2
( 1)
where r/0 = refractive index of medium, rl = refractive index of protein solution, ~, = wavelength, N A = Avogadro's number and c = particle concentration (in weight). In the case of the DynaPro-801 instrument, both the wavelength and the scattering angle are constant (780 nm and 90 °, respectively). For apoAI at 2 mg m1-1 in Trizma-HC1 5 0 m M pH = 8.0, the estimated refractive index is rl = 1.360 and the buffer alone rl0 = 1.335. In this way, the calculated geometrical factor H value is 4.80 x 1 0 -9 tool g-2 cm 2 molecule-~. Once calculated H and taking account that for small independent particles which are both optically isotropic and sufficiently small compared with the wavelength of the incident light, application of Rayleigh's theory gives: "r= HcM
(2)
where turbidity is represented as 7-, c is the particle concentration (in weight) and M is the molecular weight of protein aggregates. For a system in which the particles are aggregating in a random fashion, i.e. any size particle or aggregate combining with any other size particle or aggregate, the turbidity will increase linearly with time. Similarly, if the particles are aggregating by nucleation (in an ordered way), the turbidity will increase quadratically with time. According to Fig. 2, r changes follow an asympthothic line when Li2SO4 as a precipitant agent is used. This behavior indicates that apoAI aggregation is occurring in an ordered way and nucleation is taking place. In this way, physical information (hydrodynamic behavior) may be successfully linked with chemical information (crystal nuclei formation). 3.3. The salting-in and salting-out regions
According to the Debye-Hi.ickel theory, protein solubility has a minimum at the isoelectric point at low ionic strength, which is usually observed [17]. At
4
V.M. Bola~os-Garcia et al./Journal of Molecular Structure 440 (1998) 1-8
60
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Fig. 1. ApoAI protein solution properties measured by dynamic light scattering at different pH values: (a) diffusion coefficient, (b) hydrodynamic radius, (c) estimated molecular weight, and (d) polydispersity.
V.M. Bola~os-Garcia et al./Journal of Molecular Structure 440 (1998) 1-8
70 6O o
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U 2SO4 (moles/L.) Fig. 2. Turbidity of apoAl solution against Li2SO4 as the precipitanting agent. The tendency indicate that protein aggregation is occurring in an ordered way and nucleation events may be taking place.
low salt concentration all salts have a salting-in effect on proteins that result in a solubility increase. On the other hand, at high salt concentrations protein solubility is known to decrease and this effect is called salting-out. As shown in Fig. 3A-D, it is possible to evaluate both the salting-in and the salting-out regions for apoAI using Li2SO4 as a precipitating agent. According to the Fig. 3A, the diffusion coefficient increases with each Li2SO4 addition until its concentration reaches 0.37 M. This behavior is due to a salting-in effect, where protein solubility increases, as mentioned before. After this Li2SO4 concentration is reached, the diffusion coefficient starts to decrease since apoAI aggregation starts to take place. This change corresponds to the entry in the salting-out region. These results are in agreement with our hydrodynamic radius values. When Li2SO+ reaches 0.37 M, apoAI starts to aggregate and the hydrodynamic radius fastly increases from 12 × 10 -~8 cm to 145 × 10 -~8 cm (Fig. 3B). In the case of the estimated molecular weight, as shown in Fig. 3C, also changes from about 28 kDa to around 500 kDa. Finally, polydispersity also increases around 10 times in the
5
precipitant agent concentration range mentioned before (Fig. 3D). All of these parameters taken together, bring information related to the specific protein aggregation rates. When this aggregation occurs in a well ordered way, crystal nuclei formation might take place. These observations are related with metastable zone in Oswald-Miers phase diagrams, where nucleation phenomena takes place [20]. Once a nucleus reaches a specific size, going through the critical size, it grows spontaneously to form a macroscopic solid phase. If the rate of aggregation is stepwise controlled and diffusion maintained, the cluster will form a crystal; if not, a quick growth rate of clustering will be achieved and an amorphous precipitate will be obtained. It is well known that an optimal amount of protein is desirable for growing large enough crystals for X-ray analysis. In the case of the experimental conditions employed for dynamic light scattering experiments, the optimal protein concentration to be used depends on the molecular weight of the sample. For instance, from 0.1 for large macromolecules up to 4 mg ml -j for proteins with low molecular weight. In order to crystallize a new protein, using data coming from DLS analysis, it is possible to scale not only the concentration of protein solution but also the precipitating agent concentration to reach a suitable degree of supersaturation to provoke macroscopic crystal growth. As a consequence of this observation, crystallization trials for apoAI were made using two to three times the concentration of both protein and precipitant agent with respect to the concentration used in DLS experiments. At the beginning, several experiments were done using the same concentration employed for DLS measurements in order to initiate our crystallization trials, but extremely small crystals were obtained (Fig. 4A). Therefore, maintaining the ratio protein/precipitant agent new crystallization trials with both the hanging-drop and sitting-drop techniques were performed [21]. After 48 hours with both techniques crystal nuclei were formed when apoA] concentration was maintained between 2 and 4 mg ml i and Li2SO4 kept between 0.5-1.0 M as shown in Fig. 4B. Since these dentritic-shaped crystals obtained by DLS analysis apparently are grown by a two-dimensional mechanism, the demonstration of this assumption will be only obtained using atomic force microscopy [22].
6
V.M. Bola~os-Garcia et al./Journal of Molecular Structure 440 (1998) 1-8
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Fig. 3. ApoAI solution properties at increased Li2SO4 concentration: (a) diffusion coefficient, (b) hydrodynamic radius, (c) estimated molecular weight, and (d) polydispersity. Both salting-in and salting-out regions can be appreciated (see the text for details).
V.M. Bolaaos-Garcla et al./Journal of Molecular Structure 440 (1998) 1-8
a
7
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Fig. 4, Photograph of apoAl: (A) small crystals obtained by direct concentrations from DLS in precrystallization experiments, and (B) scaling two or three times the protein and precipitating concentration, two-dimensional dendritic-shaped crystals were obtained by the hanging-drop technique under the following conditions: protein concentration 4 mg ml-~ in Trizma 50mM pH 8.0, Li 2SO4 1.0 M, temperature 20°C. Similar morphology was observed using the sitting-drop technique.
8
V.M. Bola~os-Garcia et al./Journal of Molecular Structure 440 (1998) 1 - 8
Employing apoAI at 6 mg ml -~ and Li2SO 4 at 1.5 M, an amorphous precipitate is obtained, that might be due to the fact that an equilibrium state is quickly reached. Using the hanging-drop technique, the crystal nuclei formed are slightly bigger than using the sitting-drop technique. Although these nuclei are not large enough for X-ray crystallographic analysis, protein nucleus formation can be considered as a precrystallization step. Our next goal will be focused on growing these nuclei until they can reach a suitable size for X-ray diffraction analysis. This will be done by the crystallization method in capillary tubes as has been showed by Garcfa-Ruiz e t al. [23]. It is important to emphasize that the slow supply of molecules by diffusion causes a reduction of the protein concentration in the vicinity of a rapidly growing crystal. In this way, the reduced protein concentration prevents the formation of new nuclei and therefore, eliminates unwanted interference between crystals. Besides, crystals that grow too fast usually reach a smaller terminal size, presumably due to structural defects that can be incorporated. Consequently, the size of the crystal depends on the degree of supersaturation as well as on the kinetic pathway of nucleation and growth. In this fashion it is very important to determine the effect of several parameters such as pH, ionic strength, temperature and precipitating agent concentration on protein solubility in order to perform a rational screening for crystallization. As demonstrated here, the use of DLS can be considered a very useful technique to reach this goal. Using this technique it is possible to match physical measurements of macromolecules with their chemical properties in order to yield crystals.
Acknowledgements This study has been partially supported by D G A P A - U N A M (Grant IN-201294). Vfctor Bolafios was partially supported by PADEP, UNAM (no. 030381). Abel Moreno acknowledges finantial support from C O N A C y T in Programa de Repatriaci6n
de Cientfficos Mexicanos. This is a contribution (1597) from Instituto de Qufmica, UNAM.
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