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Groel crystal growth and characterization
BioSystems 94 (2008) 223–227
Contents lists available at ScienceDirect
BioSystems journal homepage: www.elsevier.com/locate/biosystems
Groel crystal growth and characterization E. Pechkova, S. Tripathi, R. Spera, C. Nicolini ∗ Nanoworld Institute, CIRSDNNOB-University of Genova and Fondazione Elba, Rome, Italy
a r t i c l e
i n f o
Article history: Received 14 May 2008 Accepted 31 May 2008 Keywords: Ribosomal proteins GroEL LB protein thin film Crystallization
a b s t r a c t Single crystals of ribosomal proteins obtained for the first time by Langmuir–Blodgett (LB) nanotemplate confirm earlier findings (Pechkova et al., 2008), pointing to a new generation of bionanomaterials of unique structure–function relationship. The ribosomal protein phage GroEL was overexpressed in E. coli. Since these protein’s samples have some difficulties by classical vapour diffusion method to yield optimal diffraction quality and order (GroEL), the LB nanotemplate method has been applied and compared to the classical method. With the thin film nanotemplate method large phage GroEL crystals appeared in few days and were subsequently characterized by MALDI-TOF Mass Spectroscopy and by a very preliminary X-ray diffraction. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Since some protein have some difficulties to be crystallized by classical crystallization method, the recently developed Langmuir–Blodgett nanotemplate crystallization method (Pechkova and Nicolini, 2004) can be applied. This method in principle could allow also to improve the crystal quality and radiation stability (Pechkova et al., 2004). Phage growth E large (GroEL) in the E. coli cytoplasm, the first member of hsp60 family to be identified, was recognized as a chromosomally encoded product whose deficiency resulted in defective morphogenesis of bacteriophage and T4 head structures and T5 tail structures, suggesting a role in protein assembly (Horwich and Willison, 1993). Hsp60 is a well characterized chaperone mainly localized in mitochondria of eukaryotic cells (Cheng et al., 1989; Martin et al., 1993; Soltys and Gupta, 1996). Hsp60, also known as phage growth E large (GroEL) in bacteria, is involved in the folding and assembly of polypeptide chains into oligomeric complexes. Despite the protein was widely studied (Fersht, 1996), in some conditions the crystal possess low diffraction quality and appear to be quite disordered (M. Garber, personal communication). Indeed, it is sometimes difficult and/or time-consuming to obtain crystals of suitable size for single crystal X-ray diffraction measurements. Whenever proteins are difficult to crystallize and to optimally diffract, powder diffraction technique serves indeed as an important tool to predict the space group of protein crystals and can give us a range of complementary
∗ Corresponding author at: Nanoworld Institute, University of Genova, Corso Europa 30, 16132 Genova, Italy. Tel.: +39 010 35338220; fax: +39 010 35338217. E-mail address: [email protected] (C. Nicolini). 0303-2647/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.biosystems.2008.05.031
information, which is difficult to get from single crystal Xray diffraction techniques (Von Dreele, 2006; Margiolaki et al., 2005). In our hands, LB nanotemplate method have shown prominent results and properties in protein crystallization in some cases, namely for human CK2 alpha kinase (Pechkova et al., 2003), Cytochrome P450scc (Nicolini and Pechkova, 2006) and lysozyme (Pechkova and Nicolini, 2001; Pechkova et al., 2005). Recently even the microcrystals of other ribosomal proteins (Pechkova et al., 2008) have been obtained and their diffraction analysis is being probed by synchrotron radiation (e.g. by microfocussed beamline, Riekel, 2000; Riekel et al., 2005) or at the powder diffraction beamlines (Margiolaki et al., 2005), Peaks obtained in powder diffraction analysis depend on the microstructure of materials and thus accurate unit cell information can be obtained even from poorly diffracting ribosomal proteins crystals (this issue discussed in details in the separate communication by Tripathi et al., 2008). 2. Materials and Methods 2.1. Expression and Purification Procedure The phage GroEL protein was overproduced by M. Garber group in Protein Research Institute (Puschino, Russia) in bacterial expression system. E. coli. BL21(DE3) transformed with the plasmid carrying GroEL gene was grown at 37 ◦ C to the cell density of 0.5–0.7 A600 in 2xTY medium supplemented with 100 l/ml ampicillin. Expression of GroEL was induced by IPTG adding (final concentration 1 mM), followed by 3 h culture growth at 37 ◦ C. Harvested cells (2 g) were suspended in 10 ml of buffer A (50 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 5 mM -mercaptoethanol), and sonicated. The cell debris was removed from the cell lysate by centrifugation (16,000 × g for 20 min). Ammonium sulfate powder was added to the supernatant up to 1.5 M and the protein solution was loaded onto Butyl-Toyopearl column (10 ml) preliminary equilibrated by buffer A containing 1.5 M ammonium sulfate. Ten column volumes of buffer A were used for impurities washing out. The protein was eluted by buffer A containing 200 mM NaCl and 25% ethanol. Fractions containing
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GroEL were combined, concentrated up to 20–30 mg/ml using “Vivaspin” concentrator 100 kDa and subjected to crystallization (M. Garber, personal communication).
communication). The various trials were performed in order to optimize these conditions.
2.2. Crystallization by Classical Vapour Diffusion and LB Nanotemplate Method
3. Mass Spectrometry
A thin film (Langmuir–Blodgett) LB nanotemplate method was applied (Pechkova and Nicolini, 2004). Thin protein films of GroEL were prepared by LB Teflon trough with surface pressure control system. The protein solution were spread on the air-buffer interface using the Hamilton syringe, the volume of the purified and concentrated protein was about 100 l. The protein monolayer was compressed by two Teflon barrier up to the surface pressure 20 mN/m and transferred to the cleaned glass cover slide by the modification of the Langmuir–Blodgett technique—Langmuir–Schaeffer method (horizontal lift). After that, the protein monolayer was dried in the nitrogen flux and the second monolayer was deposited onto the first one in the same way. Well-shaped isotherm (dependence of the surface pressure from the barrier position (Pechkova and Nicolini, 2003) (not shown) were obtained and more than 20 template prepared at the chosen constant surface pressure conditions. In the LB nanotemplate method, the droplet with protein solution and the crystallizing agent was placed on the thin protein film, deposited on the siliconized glass cover slide. The droplet was equilibrated against the reservoir solution contains the crystallizing agent with the concentration twice as much as in the droplet, as it is usually done by classical vapour diffusion method. For crystallization the sample of GroEL with concentration 15 mg/ml was prepared in the following buffer: 50 mM Tris–HCl pH 8, 200 mM NaCl (buffer A). The preliminary screening for crystallization conditions was carried out using classical hanging drop vapour diffusion technique. Initially, crystalline precipitation were observed in solution containing 100 mM Hepes, pH 7.5, 10% PEG 8000, 8% ethylene glycol (Hampton research Screen II No. 37) (M. Garber, personal
We used matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS, Bruker) to monitor the purity of proteins solutions of crystallographic interest. These solutions had been preventively dialyzed and/or the proteins had been precipitated in a solution of trichloroacetic acid to eliminate any trace of glycerol, salt and detergent that prevent the protein ionization. The protein samples were diluted in a 0.1% (v/v) TFA solution. The matrix used for the mass spectrometric analysis was a saturated solution of acid (␣-cyano-4-hydroxycinnamic acid for light proteins and sinapinic acid for heavy proteins, Bruker Daltonics) dissolved in 2/3 of 0.1% (v/v) TFA and 1/3 of acetonitrile. 1.5 l of matrix solution was mixed with 1.5 l of sample, then 1 l of this mixture is spotted onto a suitable aluminium plate and airdried. MALDI-MS spectra were acquired in positive ion linear mode using an Autoflex mass spectrometer (Bruker Daltonics) externally calibrated using a solution of protein of known masses’ resulting in a mass accuracy <100 ppm for intact proteins.
Fig. 1. (A) GroEL crystals, obtained by LB nanotemplate method under the light microscope (crystals dimensions are about 200 m × 200 m × 100 m); (B) GroEL needle microcrystals, obtained by LB nanotemplate method under the light microscope (crystals dimensions are about 100 m × 10 m × 10 m); (C) one of the needle microcrystal mounted in the cryoloop, bar corresponds to 50 m); (D) GroEL crystals obtained by classical hanging drop method (crystals dimensions are about 50 m × 5 m × 5 m).
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225
It have been analysed only intact protein or dissolve crystals; a trypsin digestion was occasionally performed for fingerprint analysis. The results are consistent and satisfactory, confirming the high purity level of each solution.
ware. The principle of the Rietveld method (Rietveld, 1969) is to minimise a function M which represents the difference between a calculated profile y(calc) and the observed data y(obs), namely:
4. Preliminary Diffraction Analysis
M=
Preliminary diffraction data were collected at a temperature of 100 K. Crystals were removed from the mother liquor and frozen in a nitrogen stream using cryoprotectant, 50% (v/v) of Paraton-N (Hampton Research) in distilled water. The beam size was 5 m × 5 m; the wavelength used was 0.9755 Å and the crystal-to-detector distance was 130 mm. One nanotemplate crystal was used (Nicolini and Pechkova, 2006; Pechkova et al., 2008) to collect the complete data sets at the microfocus beamline ID13 at the ESRF (Riekel, 2004). The beam size was 5 m × 5 m; the wavelength used was 0.9755 Å and the crystal-to-detector distance was 130 mm. Crystals diffracted to a maximum resolution of 1.7 Å. By analogy with the processing of data from lysozyme powder crystals shown in Von Dreele (2006) (http://www.aps.anl.gov/Science/ Future/Workshops/Biological Crystallography/Presentations/Von% 20Dreele.pdf), the diffracting ring patterns obtained on the above crystals were subsequently studied using the FIT2D soft-
i
Wi yiobs −
1 calc y c i
2
where Wi is a statistical weight and c is an overall scale factor such that yc = cy. The GUI software EXPGUI is used for the refinement process. The work is still largely in progress and needed further experimentation. 5. Results and Discussion We obtain the GroEL protein crystals with LB nanotemplate in order to attempt their further improvement, being aware that the crystals of this protein system have been widely and successfully characterized worldwide down to the atomic scale (i.e., Chaudhry et al., 2004; Zahn et al., 1996). Two optimized crystallization conditions were defined:
Fig. 2. Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS spectra of the dissolved crystals of: GroEL. The protein mass is 61.6 ± 0.6 kDa, compatible with the nominal mass. The two peaks are assigned as [m]+ for m/z = 61646 and [m]2+ (double charge) for m/z = 30754.
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1. The mixture, containing 5 (l of the GroEL (15 mg/ml) in buffer A with 5 (l AMP and 1 (l PEG 8000 10% (m/v) Droplets (3 l) of above mixture were placed on the nanofilm (2 layers) equilibrated against 1000 ml PEG 8000 40% (m/v) reservoir with solution with template at 20 ◦ C. Rather big single crystals up to 200 m × 200 m × 100 m were obtained after few days (Fig. 1A). It is worth to notice that classical hanging drop method can also result in growth of rather big GroEL crystal (M. Garber, personal communications), but with low diffraction quality. 2. The mixture, containing 5 (l of the GroEL (15 mg/ml) in buffer A with 5 (l AMP and 1 (l PEG 6000 10% (m/v) Droplets (3 l) of above mixture were placed on the nanofilm (2 layers) was equilibrated against 1000 ml PEG 6000 40% (m/v) reservoir with solution with template at 20 ◦ C. Needle crystals (100 m × 10 m × 10 m) appears after 1 week, clearly visible under a light microscope (Fig. 1B). It was possible to mount these crystals in the nylon cryoloop (Fig. 1C). In similar conditions, needle crystals was observed also by classical hanging drop vapour diffusion method, but significantly smaller (50 m × 5 m × 5 m), than those obtained by LB nanotemplate (Fig. 1C). All obtained crystals grown were dissolved in buffer A and were analysed by mass spectroscopy. As the crystals have the different shape and morphology, the mass spectroscopy analysis is useful to confirms that the obtained crystals are indeed the crystals of the protein of interest and not the salt or other precipitant crystals, as can occur in the crystallization trials observation. This analysis confirm that the crystals were indeed composed GroEL, molecular weight 60 kDa (Fig. 2). The GroEL crystals grown both by classical and LB nanotemplate method was mounted in the cryoloops and analysed by synchrotron radiation (Fig. 1D). So far, the protein crystals of this dimensions were useless for X-ray diffraction experiments for protein 3D atomic structure resolution, also because of the radiation damage issue. LB nanotemplate method, already been successful in the crystallization of proteins so far impossible with classical methods (Pechkova and Nicolini, 2004) appears indeed to be able to produce also protein crystals more stable to synchrotron radiation (Pechkova et al., 2004, 2005). With the recent advances in micro- and nanofocusing of synchrotron X-ray beam (Riekel, 2000; Riekel et al., 2005) (see also http://www.esrf.eu/UsersAndScience/ Experiments/SCMatter/ID13/nanofocus), even very small crystals can thereby be used for the X-ray diffraction experiments. The obtained GroEL microcrystals have been then submitted to a very preliminary diffraction analysis by synchrotron radiation facility at the ID13 microfocussed beamline (Riekel, 2000) and X-ray diffraction images were obtained from the polycrystalline materials resulting from the above crystallization experiments. The crystals obtained by classical method appeared to be quite disordered, and do not give any diffraction signal, while crystals grown by LB nanotemplate method, both large (200 m × 100 m × 100 m) and small (20 m × 10 m × 10 m) showed the diffracting ring patterns, due to superposition of individual diffraction from a large number of randomly oriented crystallites likely present in this powder-like diffraction (Fig. 3). The GroEL crystal was nearly invisible after being cryoprotected before the exposure to the synchrotron radiation which called for the need of Paraton-N (Hampton Research, a cryoprotectant). As a consequence a blind search was carried out in an area of 20 m × 55 m with diffraction being acquired at 5 m increment within the loop; it was the map resulting from this systematic blind search (Fig. 3) that allows to localize regions showing wide rings near the center in the limited area of 20 m × 55 m being scanned corresponding to a portion of the crystal shown in Fig. 1A.
Fig. 3. Map of diffraction patterns along the GroEL LB crystal shown in Fig. 1A which has been cryoprotected with Paraton-N before the exposure to the synchrotron radiation. This map results from a blind search in an area of 20 m × 55 m with 5 m increment and allow to identify the crystal regions displaying rings in the patterns.
6. Conclusions In the present report, the improved LB-based crystallization procedure was introduced for phage GroEL protein overproduced in E. coli. In our hands, the GroEL protein crystal was grown better with LB nanotemplate: (a) the crystals is larger, than those grown in the same conditions by classical method; (b) in the presence of LB template, the protein is crystallizing in two different crystallization conditions while by the classical method only in one. As we do not report here the single crystal X-ray diffraction data with resulting protein structure, the crystals were proven to be indeed GroEL crystals by mass spectrometry analysis. The poor quality of the obtained diffraction data does not presently allow to extract the structural information, e.g. the unit cell identification, calling for further experimentation and further development in powder-like diffraction technique. Acknowledgements This work was supported by a FIRB-MIUR grant on Proteomics to the Nanoworld Institute of University of Genoa and by a PNRMIUR grant on Biocatalysis to the University of Genoa and to the Fondazione Elba. We are particularly grateful to Marina Garber, Uliana Tin and Vadim Mesyanzhino from the Institute of Protein Research RAS, Pushchino (Russia) for providing the GroEL proteins and the procedure for their crystallization by classical method and to Udo Bläsi of Vienna Biocenter (Austria) for providing the clones.
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References Chaudhry, C., Horwich, A.L., Brunger, A.T., Adams, P.D., 2004. Exploring the structural dynamics of the E. coli chaperonin GroEL using translation–libration-screw crystallographic refinement of intermediate states. J. Mol. Biol. 342, 229–245. Cheng, M.Y., Hartl, F.U., Martin, J., Pollock, R.A., Kalousek, F., Neupert, W., Hallberg, E.M., Hallberg, R.L., Horwich, A.L., 1989. Nature 337, 620–625. Fersht, A.R., 1996. Chaperone activity and structure of monomeric polypeptide binding domains of GroEL. Proc. Natl. Acad. Sci. U.S.A. 93, 15024–15029. Horwich, A.L., Willison, K.R., 1993. Protein folding in the cell: functions of two families of molecular chaperone, hsp 60 and TF55-TCP1. Philos. Trans.: Biol. Sci. 339 (1289), 313–326 (Molecular Chaperones). Margiolaki, M.I., Wright, J.P., Fitch, A.N., Fox, G.C., Von Dreele, R.B., 2005. Acta Cryst. D61, 423–432. Martin, J., Mayhew, M., Langer, T., Hartl, U., 1993. The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding. Nature 366, 228–233. Nicolini, C., Pechkova, E., 2006. Structure and growth of ultrasmall protein microcrystals by synchrotron radiation. I. GISAXS and diffraction of P450scc. J. Cell. Biochem. 97, 544–552. Pechkova, E., Nicolini, C., 2001. Accelerated protein crystal growth onto the protein thin film. J. Cryst. Growth 231, 599–602. Pechkova and Nicolini, 2003. Proteomics and Nanocrystallography, Kluwer Academic Plenum Publishers, pp. 1–212. Pechkova, E., Nicolini, C., 2004. Protein nanocrystallography: a new approach to structural proteomics. Trends Biotechnol. 22, 117–122.
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Pechkova, E., Zanotti, G., Nicolini, C., 2003. Three-dimensional atomic structure of a catalytic subunit mutant of human protein kinase CK2. Acta Crystallogr. D 59, 2133–2139. Pechkova, E., Tropiano, G., Riekel, C., Nicolini, C., 2004. Radiation stability of protein crystals grown by nanostructured templates: synchrotron microfocus analysis. Spectrochim. Acta B 59, 1687–1693. Pechkova, E., Vasile, F., Spera, R., Nicolini, C., 2005. Protein nanocrystallography: growth mechanism and atomic structure of crystal induced by nanotemplates. J. Synchrotron Radiat. 12, 772–778. Pechkova, E., Vasile, F., Spera, R., Nicolini, C., 2008. Crystallization of alpha and beta subunits of IF2 translation initiation factor from Archaebacteria Sulfolobus Solfataricus. J. Cryst. Growth 310, 3767–3770. Rietveld, H., 1969. J. Appl. Crystallogr. 2, 65–71. Riekel, C., 2000. New avenues in X-ray microbeam experiments. Rep. Prog. Phys. 63, 233–262. Riekel, C., Burghammer, M., Schertler, G.F.X., 2005. Protein crystallography microdiffraction. Curr. Opin. Struct. Biol. 15, 556–562. Soltys, B.J., Gupta, R.S., 1996. Exp. Cell Res. 222, 16–27. Tripathi, S., Pechkova, E., Nicolini, 2008. Powder diffraction analysis for initiation factor subunits and p450scc cytochrome. J. Synchrotron Radiat., still under consideration needing extensive revision. Von Dreele, R.B., 2006. Rev. Mineral. Geochem. 63, 81–98. Zahn, R., Buckle, A. M., Perrett, S., Johnson, C. M., Corrales,. F. J., Golbik, R. & Fersht, A. R. (1996). Proc. Natl. Acad. Sci. USA. 93, 15024–15029.
Contents lists available at ScienceDirect
BioSystems journal homepage: www.elsevier.com/locate/biosystems
Groel crystal growth and characterization E. Pechkova, S. Tripathi, R. Spera, C. Nicolini ∗ Nanoworld Institute, CIRSDNNOB-University of Genova and Fondazione Elba, Rome, Italy
a r t i c l e
i n f o
Article history: Received 14 May 2008 Accepted 31 May 2008 Keywords: Ribosomal proteins GroEL LB protein thin film Crystallization
a b s t r a c t Single crystals of ribosomal proteins obtained for the first time by Langmuir–Blodgett (LB) nanotemplate confirm earlier findings (Pechkova et al., 2008), pointing to a new generation of bionanomaterials of unique structure–function relationship. The ribosomal protein phage GroEL was overexpressed in E. coli. Since these protein’s samples have some difficulties by classical vapour diffusion method to yield optimal diffraction quality and order (GroEL), the LB nanotemplate method has been applied and compared to the classical method. With the thin film nanotemplate method large phage GroEL crystals appeared in few days and were subsequently characterized by MALDI-TOF Mass Spectroscopy and by a very preliminary X-ray diffraction. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Since some protein have some difficulties to be crystallized by classical crystallization method, the recently developed Langmuir–Blodgett nanotemplate crystallization method (Pechkova and Nicolini, 2004) can be applied. This method in principle could allow also to improve the crystal quality and radiation stability (Pechkova et al., 2004). Phage growth E large (GroEL) in the E. coli cytoplasm, the first member of hsp60 family to be identified, was recognized as a chromosomally encoded product whose deficiency resulted in defective morphogenesis of bacteriophage and T4 head structures and T5 tail structures, suggesting a role in protein assembly (Horwich and Willison, 1993). Hsp60 is a well characterized chaperone mainly localized in mitochondria of eukaryotic cells (Cheng et al., 1989; Martin et al., 1993; Soltys and Gupta, 1996). Hsp60, also known as phage growth E large (GroEL) in bacteria, is involved in the folding and assembly of polypeptide chains into oligomeric complexes. Despite the protein was widely studied (Fersht, 1996), in some conditions the crystal possess low diffraction quality and appear to be quite disordered (M. Garber, personal communication). Indeed, it is sometimes difficult and/or time-consuming to obtain crystals of suitable size for single crystal X-ray diffraction measurements. Whenever proteins are difficult to crystallize and to optimally diffract, powder diffraction technique serves indeed as an important tool to predict the space group of protein crystals and can give us a range of complementary
∗ Corresponding author at: Nanoworld Institute, University of Genova, Corso Europa 30, 16132 Genova, Italy. Tel.: +39 010 35338220; fax: +39 010 35338217. E-mail address: [email protected] (C. Nicolini). 0303-2647/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.biosystems.2008.05.031
information, which is difficult to get from single crystal Xray diffraction techniques (Von Dreele, 2006; Margiolaki et al., 2005). In our hands, LB nanotemplate method have shown prominent results and properties in protein crystallization in some cases, namely for human CK2 alpha kinase (Pechkova et al., 2003), Cytochrome P450scc (Nicolini and Pechkova, 2006) and lysozyme (Pechkova and Nicolini, 2001; Pechkova et al., 2005). Recently even the microcrystals of other ribosomal proteins (Pechkova et al., 2008) have been obtained and their diffraction analysis is being probed by synchrotron radiation (e.g. by microfocussed beamline, Riekel, 2000; Riekel et al., 2005) or at the powder diffraction beamlines (Margiolaki et al., 2005), Peaks obtained in powder diffraction analysis depend on the microstructure of materials and thus accurate unit cell information can be obtained even from poorly diffracting ribosomal proteins crystals (this issue discussed in details in the separate communication by Tripathi et al., 2008). 2. Materials and Methods 2.1. Expression and Purification Procedure The phage GroEL protein was overproduced by M. Garber group in Protein Research Institute (Puschino, Russia) in bacterial expression system. E. coli. BL21(DE3) transformed with the plasmid carrying GroEL gene was grown at 37 ◦ C to the cell density of 0.5–0.7 A600 in 2xTY medium supplemented with 100 l/ml ampicillin. Expression of GroEL was induced by IPTG adding (final concentration 1 mM), followed by 3 h culture growth at 37 ◦ C. Harvested cells (2 g) were suspended in 10 ml of buffer A (50 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 5 mM -mercaptoethanol), and sonicated. The cell debris was removed from the cell lysate by centrifugation (16,000 × g for 20 min). Ammonium sulfate powder was added to the supernatant up to 1.5 M and the protein solution was loaded onto Butyl-Toyopearl column (10 ml) preliminary equilibrated by buffer A containing 1.5 M ammonium sulfate. Ten column volumes of buffer A were used for impurities washing out. The protein was eluted by buffer A containing 200 mM NaCl and 25% ethanol. Fractions containing
224
E. Pechkova et al. / BioSystems 94 (2008) 223–227
GroEL were combined, concentrated up to 20–30 mg/ml using “Vivaspin” concentrator 100 kDa and subjected to crystallization (M. Garber, personal communication).
communication). The various trials were performed in order to optimize these conditions.
2.2. Crystallization by Classical Vapour Diffusion and LB Nanotemplate Method
3. Mass Spectrometry
A thin film (Langmuir–Blodgett) LB nanotemplate method was applied (Pechkova and Nicolini, 2004). Thin protein films of GroEL were prepared by LB Teflon trough with surface pressure control system. The protein solution were spread on the air-buffer interface using the Hamilton syringe, the volume of the purified and concentrated protein was about 100 l. The protein monolayer was compressed by two Teflon barrier up to the surface pressure 20 mN/m and transferred to the cleaned glass cover slide by the modification of the Langmuir–Blodgett technique—Langmuir–Schaeffer method (horizontal lift). After that, the protein monolayer was dried in the nitrogen flux and the second monolayer was deposited onto the first one in the same way. Well-shaped isotherm (dependence of the surface pressure from the barrier position (Pechkova and Nicolini, 2003) (not shown) were obtained and more than 20 template prepared at the chosen constant surface pressure conditions. In the LB nanotemplate method, the droplet with protein solution and the crystallizing agent was placed on the thin protein film, deposited on the siliconized glass cover slide. The droplet was equilibrated against the reservoir solution contains the crystallizing agent with the concentration twice as much as in the droplet, as it is usually done by classical vapour diffusion method. For crystallization the sample of GroEL with concentration 15 mg/ml was prepared in the following buffer: 50 mM Tris–HCl pH 8, 200 mM NaCl (buffer A). The preliminary screening for crystallization conditions was carried out using classical hanging drop vapour diffusion technique. Initially, crystalline precipitation were observed in solution containing 100 mM Hepes, pH 7.5, 10% PEG 8000, 8% ethylene glycol (Hampton research Screen II No. 37) (M. Garber, personal
We used matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS, Bruker) to monitor the purity of proteins solutions of crystallographic interest. These solutions had been preventively dialyzed and/or the proteins had been precipitated in a solution of trichloroacetic acid to eliminate any trace of glycerol, salt and detergent that prevent the protein ionization. The protein samples were diluted in a 0.1% (v/v) TFA solution. The matrix used for the mass spectrometric analysis was a saturated solution of acid (␣-cyano-4-hydroxycinnamic acid for light proteins and sinapinic acid for heavy proteins, Bruker Daltonics) dissolved in 2/3 of 0.1% (v/v) TFA and 1/3 of acetonitrile. 1.5 l of matrix solution was mixed with 1.5 l of sample, then 1 l of this mixture is spotted onto a suitable aluminium plate and airdried. MALDI-MS spectra were acquired in positive ion linear mode using an Autoflex mass spectrometer (Bruker Daltonics) externally calibrated using a solution of protein of known masses’ resulting in a mass accuracy <100 ppm for intact proteins.
Fig. 1. (A) GroEL crystals, obtained by LB nanotemplate method under the light microscope (crystals dimensions are about 200 m × 200 m × 100 m); (B) GroEL needle microcrystals, obtained by LB nanotemplate method under the light microscope (crystals dimensions are about 100 m × 10 m × 10 m); (C) one of the needle microcrystal mounted in the cryoloop, bar corresponds to 50 m); (D) GroEL crystals obtained by classical hanging drop method (crystals dimensions are about 50 m × 5 m × 5 m).
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225
It have been analysed only intact protein or dissolve crystals; a trypsin digestion was occasionally performed for fingerprint analysis. The results are consistent and satisfactory, confirming the high purity level of each solution.
ware. The principle of the Rietveld method (Rietveld, 1969) is to minimise a function M which represents the difference between a calculated profile y(calc) and the observed data y(obs), namely:
4. Preliminary Diffraction Analysis
M=
Preliminary diffraction data were collected at a temperature of 100 K. Crystals were removed from the mother liquor and frozen in a nitrogen stream using cryoprotectant, 50% (v/v) of Paraton-N (Hampton Research) in distilled water. The beam size was 5 m × 5 m; the wavelength used was 0.9755 Å and the crystal-to-detector distance was 130 mm. One nanotemplate crystal was used (Nicolini and Pechkova, 2006; Pechkova et al., 2008) to collect the complete data sets at the microfocus beamline ID13 at the ESRF (Riekel, 2004). The beam size was 5 m × 5 m; the wavelength used was 0.9755 Å and the crystal-to-detector distance was 130 mm. Crystals diffracted to a maximum resolution of 1.7 Å. By analogy with the processing of data from lysozyme powder crystals shown in Von Dreele (2006) (http://www.aps.anl.gov/Science/ Future/Workshops/Biological Crystallography/Presentations/Von% 20Dreele.pdf), the diffracting ring patterns obtained on the above crystals were subsequently studied using the FIT2D soft-
i
Wi yiobs −
1 calc y c i
2
where Wi is a statistical weight and c is an overall scale factor such that yc = cy. The GUI software EXPGUI is used for the refinement process. The work is still largely in progress and needed further experimentation. 5. Results and Discussion We obtain the GroEL protein crystals with LB nanotemplate in order to attempt their further improvement, being aware that the crystals of this protein system have been widely and successfully characterized worldwide down to the atomic scale (i.e., Chaudhry et al., 2004; Zahn et al., 1996). Two optimized crystallization conditions were defined:
Fig. 2. Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS spectra of the dissolved crystals of: GroEL. The protein mass is 61.6 ± 0.6 kDa, compatible with the nominal mass. The two peaks are assigned as [m]+ for m/z = 61646 and [m]2+ (double charge) for m/z = 30754.
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1. The mixture, containing 5 (l of the GroEL (15 mg/ml) in buffer A with 5 (l AMP and 1 (l PEG 8000 10% (m/v) Droplets (3 l) of above mixture were placed on the nanofilm (2 layers) equilibrated against 1000 ml PEG 8000 40% (m/v) reservoir with solution with template at 20 ◦ C. Rather big single crystals up to 200 m × 200 m × 100 m were obtained after few days (Fig. 1A). It is worth to notice that classical hanging drop method can also result in growth of rather big GroEL crystal (M. Garber, personal communications), but with low diffraction quality. 2. The mixture, containing 5 (l of the GroEL (15 mg/ml) in buffer A with 5 (l AMP and 1 (l PEG 6000 10% (m/v) Droplets (3 l) of above mixture were placed on the nanofilm (2 layers) was equilibrated against 1000 ml PEG 6000 40% (m/v) reservoir with solution with template at 20 ◦ C. Needle crystals (100 m × 10 m × 10 m) appears after 1 week, clearly visible under a light microscope (Fig. 1B). It was possible to mount these crystals in the nylon cryoloop (Fig. 1C). In similar conditions, needle crystals was observed also by classical hanging drop vapour diffusion method, but significantly smaller (50 m × 5 m × 5 m), than those obtained by LB nanotemplate (Fig. 1C). All obtained crystals grown were dissolved in buffer A and were analysed by mass spectroscopy. As the crystals have the different shape and morphology, the mass spectroscopy analysis is useful to confirms that the obtained crystals are indeed the crystals of the protein of interest and not the salt or other precipitant crystals, as can occur in the crystallization trials observation. This analysis confirm that the crystals were indeed composed GroEL, molecular weight 60 kDa (Fig. 2). The GroEL crystals grown both by classical and LB nanotemplate method was mounted in the cryoloops and analysed by synchrotron radiation (Fig. 1D). So far, the protein crystals of this dimensions were useless for X-ray diffraction experiments for protein 3D atomic structure resolution, also because of the radiation damage issue. LB nanotemplate method, already been successful in the crystallization of proteins so far impossible with classical methods (Pechkova and Nicolini, 2004) appears indeed to be able to produce also protein crystals more stable to synchrotron radiation (Pechkova et al., 2004, 2005). With the recent advances in micro- and nanofocusing of synchrotron X-ray beam (Riekel, 2000; Riekel et al., 2005) (see also http://www.esrf.eu/UsersAndScience/ Experiments/SCMatter/ID13/nanofocus), even very small crystals can thereby be used for the X-ray diffraction experiments. The obtained GroEL microcrystals have been then submitted to a very preliminary diffraction analysis by synchrotron radiation facility at the ID13 microfocussed beamline (Riekel, 2000) and X-ray diffraction images were obtained from the polycrystalline materials resulting from the above crystallization experiments. The crystals obtained by classical method appeared to be quite disordered, and do not give any diffraction signal, while crystals grown by LB nanotemplate method, both large (200 m × 100 m × 100 m) and small (20 m × 10 m × 10 m) showed the diffracting ring patterns, due to superposition of individual diffraction from a large number of randomly oriented crystallites likely present in this powder-like diffraction (Fig. 3). The GroEL crystal was nearly invisible after being cryoprotected before the exposure to the synchrotron radiation which called for the need of Paraton-N (Hampton Research, a cryoprotectant). As a consequence a blind search was carried out in an area of 20 m × 55 m with diffraction being acquired at 5 m increment within the loop; it was the map resulting from this systematic blind search (Fig. 3) that allows to localize regions showing wide rings near the center in the limited area of 20 m × 55 m being scanned corresponding to a portion of the crystal shown in Fig. 1A.
Fig. 3. Map of diffraction patterns along the GroEL LB crystal shown in Fig. 1A which has been cryoprotected with Paraton-N before the exposure to the synchrotron radiation. This map results from a blind search in an area of 20 m × 55 m with 5 m increment and allow to identify the crystal regions displaying rings in the patterns.
6. Conclusions In the present report, the improved LB-based crystallization procedure was introduced for phage GroEL protein overproduced in E. coli. In our hands, the GroEL protein crystal was grown better with LB nanotemplate: (a) the crystals is larger, than those grown in the same conditions by classical method; (b) in the presence of LB template, the protein is crystallizing in two different crystallization conditions while by the classical method only in one. As we do not report here the single crystal X-ray diffraction data with resulting protein structure, the crystals were proven to be indeed GroEL crystals by mass spectrometry analysis. The poor quality of the obtained diffraction data does not presently allow to extract the structural information, e.g. the unit cell identification, calling for further experimentation and further development in powder-like diffraction technique. Acknowledgements This work was supported by a FIRB-MIUR grant on Proteomics to the Nanoworld Institute of University of Genoa and by a PNRMIUR grant on Biocatalysis to the University of Genoa and to the Fondazione Elba. We are particularly grateful to Marina Garber, Uliana Tin and Vadim Mesyanzhino from the Institute of Protein Research RAS, Pushchino (Russia) for providing the GroEL proteins and the procedure for their crystallization by classical method and to Udo Bläsi of Vienna Biocenter (Austria) for providing the clones.
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