2. Protein crystalliza1on
Supramolecular Hydrogels for Protein Crystalliza1on Mª Teresa Conejero-‐Muriela, Estela Pineda-‐Molinaa, Mónica Moral Muñoz b, Juan de Dios García-‐López Duránb, Luis
Álvarez de Cienfuegosc, Juan J. Díaz-‐Mochónd, e and José A. Gaviraa.
a Laboratorio de Estudios Cristalográficos InsLtuto Andaluz Ciencias de la Tierra, Avda de las Palmeras, 4, Armilla, 18100, Granada, Spain. b Dpto. de Física Aplicada, c Dpto. de Química Orgánica, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain. d Dpto. de Química FarmacéuLca y Orgánica, Facultad de Farmacia, Universidad de Granada, 18071 Granada, Spain. e GENYO. Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government, PTS Granada, Avenida de la Ilustración 114, 18016 Granada, Spain
Email: [email protected]
3. Results
Acknowledgements This work was supported by project BIO2010-‐16800 and the “Factoría de Cristalización”, CONSOLIDER INGENIO-‐2010 from the Ministerio de Ciencia e Innovación, Spain. MTCM thanks CSIC for her JAE-Pre research contract. EPM is supported by a `Ramón y Cajal´ research contract (MINECO). JJD M benefits from Ministerio de Economia y CompeLLvidad "Ramon y
Cajal Fellowoship" . !
References [1]. a) Terech, P.; Weiss, R. G. Chem. Rev. 1997, 97, 3133. b) George, M.; Weiss, R. G. Acc. Chem. Rev. 2006, 39, 489. [2]. a) Piepenbrock, M.-‐O. M.; Lloyd, G. O.; Clarke, N., Steed, J. W. Chem. Rev. 2010, 110, 1960. b) Maeda, H. Chem. Eur. J. 2008, 36, 11274. [3]. Reches, M.; Gazit, E. Science 2003, 300, 625. [4]. a) Sangeetha, N. M.; Maitra, U. Chem. Soc. Rev. 2005, 34, 821. b) Hirst, A. R.; Escuder, B.; Miravet, J. F.; Smith, D. K. Angew. Chem. Int. Ed. 2008, 47, 8002. c) Escuder, B.; Rodríguez-‐Llansola, F.; Miravet, J. F. New J. Chem. 2010, 34, 1044. d) Steed, J. W. Chem. Soc. Rev. 2010, 39, 3686. e) Steed, J. W. Chem. Commun., 2011, 47, 1379. [5]. Antara Dasgupta, Julfikar Hassan Mondal and DebapraLm Das, RSC Adv., 2013, 3, 9117–9149. [6]. a) Lorber, B.; Sauter, C.; Theobald-‐Dietrich, A.; Moreno, A.; Schellenberger, P.; Robert, M.-‐C.; Capelle, B.; Sanglier, S.; PoLer, N.; Giege, R. Prog Biophys Mol Biol 2009, 101, (1), 13-‐25. b) Gavira, J. A.; Van Driessche, A. E. S.; Garcia-‐Ruiz, J.-‐M., Crystal Growth & Design 2013, 13, (6), 2522-‐2529. [7]. Otálora F, Gavira JA, Ng JD, García-‐Ruiz JM. Prog Biophys Mol Biol. 2009 Nov;101(1-‐3):26-‐37.
1. Introduc1on Gels are systems with a high proporLon of liquid but behave as solids with viscoelasLc properLes. Recently, novel gels based on low molecular weight gelators (LMWGs) have been developed and studied [1]. LMWGs are capable of self-‐assemble by secondary forces to give 3D supramolecular networks of nanofibers that immobilize the liquid. Supramolecular gels have inherent properLes derived from the low molecular weight and weak interacLons of the monomers such as reversibility [2] and well defined 2D structures [3]. Thus, it has allowed the development of gels with very specific and parLcular properLes that have found applicaLons in a great variety of fields from medicine to electronics [4]. PolypepLdes Supramolecular Gel (PSG) belongs to this group of LMWGs adding to their general characterisLcs a high level of biocompaLbility and tunable interacLon properLes [5]. In protein crystallizaLon the use of gels of different nature has also been widely studied and its benefits have been highlighted at different levels, i.e. the growth: improving the crystal quality or controlling the morphology; the nucleaLon: inducing or inhibiLng the nucleaLon as a funcLon of the gel nature; the crystal protecLon: prevenLng osmoLc shock, avoiding the use of cryo-‐protectants, etc [6 and references herein]. We have synthesized and characterized a family of LMW-‐Hydrogelators (HG2.6) based on self-‐assembling of small polypepLdes formed in pure water. Due to its parLcular properLes we have explored the use of PSG on the crystallizaLon of two model proteins, lysozyme (HEWL) and glucose isomerase and two target proteins, Tcm16 and TodT. DiffracLon data of these four proteins have been compared with those obtained in agarose gel.
1. Hydrogel characteriza1on 2. Protein crystalliza1on Lysozyme Glucose isomerase Tcm16
1. Gel characteriza1on
Rheometer Transmission Electron Microscopy
Circular Dichroism
Crystalliza1on method
Protein concentra1on
(mg/ml)
Precipitant
Lysozyme Two layer (2L) counter difussion
140-‐220 3.5-‐10% NaCl
Glucose isomerase Batch 100-‐140 30%PEG 4K, 0.1M Tris pH 8.5, 0.2M MgCl2
Tcm16 Counter difussion-‐GCB
20-‐40 10mM Tris pH 8.5, 1M ammonium sulphate
TodT Batch 10-‐14 10% Isopropanol, 20%PEG 4K, 0.1M
Hepes pH7.5
Ini1al protein concentra1on
(mg/ml)
Protein concentra1on in gel (mg/ml)
Precipitant
Lysozyme 200 140 6% NaCl
Glucose isomerase
110 100 30%PEG 4K, 0.1M Tris pH 8.5, 0.2M MgCl2
Tcm16 28 20 10mM Tris pH 8.5, 1M ammonium sulphate
TodT 12 9,5 10% Isopropanol, 20%PEG 4K, 0.1M Hepes
pH7.5
2. Experimental methods
RAMC 2013
Sunday, 08 September 2013 -‐ Wednesday, 11 September 2013
Table 1. IniLal crystallizaLon experiments were performed to find the best crystallizaLon condiLons for each protein.
Table 2. CrystallizaLon condiLons used in agarose gel and HG2.6.
Rheology
0.1 1 10 1000.01
0.1
1
10
100
1000
10000
G' /
G''
(Pa)
σ (Pa)
G' (2,6) - 2 mM G'' (2,6) - 2 mM G' (2,6) - 3 mM G'' (2,6) - 3 mM
Fig. 2. Oscillatory rheology of HG. The values of the storage modulus (G´) and the loss modulus (G´´) exhibit a weak dependence from 0.1 to 1.0% of strain (with G´dominaLng G´´) indicaLng that HG2.6 is a hydrogel.
4. Conclusions
Fig. 3. A) Transmission electron microscopy image of HG2.6. B) Transmission electron microscopy image of negaPvely stained HG2.6. HG2.6 self-‐assembles into nanofibers from 20 nm diameters to the range of microns.
Electron microscopy A B
Circular dichroism
Fig. 4. Circular dichroism (CD) spectrum of HG2.6. It exhibits a posiLve band near 200 nm (ππ* transiLon), a negaLve band near 215 nm (nπ* transiLon), and a negaLve band near 280 nm (ππ* of benzyl groups). These peaks are consistent with the supramolecular organizaLon of pepLdes in a β-‐sheet conformaLon.
Lysozyme Glucose isomerase TodT
3. Crystal quality evalua1on
3. X-‐Ray data collec1on
A
Protein + gel
Precipitant B
Data were collected under the same condiLons, i.e: crystal to detector distance; exposure Lme; oscillaLon degree and number of images.
Fig. 5. Lysozyme crystals grown in A) D5 agarose gel and B) in HG2.6. In both cases, crystals had similar morphology. Glucose isomerase crystals grown in C) D5 agarose gel and D) in HG2.6. In both cases, crystals had similar morphology. Tcm16 crystals grown in E) D5 agarose gel and in F) HG2.6. In both cases, crystals had similar morphology. G) TodT crystallizaPon experiment in D5 agarose gel. H) TodT crystal grown in HG2.6. In this case, crystal only grew in HG2.6. From I to L it is shown a magnificaLon of each protein crystal..
A B C D E F
I J K
-‐ As expected, HG2.6 shows the typical β-‐sheet structure and behaves as a hydrogel compose by fibers of approximately 20 nm in diameter and several microns length. -‐ HG2.6 is a suitable gel to grow protein crystals as demonstrate with two model proteins (lysozyme and glucose isomerase) and two target proteins (Tcm16 and TodT). -‐ Taking into account the crystal quality indicators, it seems that lysozyme crystals grown in HG2.6 gel present higher quality than crystals grown in agarose gel. In the case of glucose isomerase, we cannot compare the data since they belong to two different space groups. More crystals need to be analysed before we can conclude that HG2.6 grown crystals are of be|er quality than those grown in agarose.
-‐ DiffracLon data from Tcm16 in agarose gel and HG2.6 were not of sufficient quality and crystal improvement is under going. However, TodT crystal was only obtained in HG2.6 and its diffracLon data will be used for Molecular Replacement.
-‐ It is also remarkable that HG2.6 also acts as cryo-‐protectant. Crystals from HG2.6 present be|er quality than crystals from agarose gel. -‐ We propose HG2.6 as a validate candidate for protein crystallizaLon.
TodT
Lysozyme, glucose isomerase and Tcm16 crystals were diffracted at 100K with and without glycerol as cryo-‐protectant. TodT crystal was diffracted with PEG 400 as cryo-‐protectant.
G H
L
Fig. 6. Lysozyme diffrac5on results. A) Lysozyme diffracLon pa|ern. B) Plot of I/σ against resoluLon to compare lysozyme crystal quality grown in agarose gel and HG2.6 and collected with and without cryo-‐protectant at 100K. The table resumes the X-‐Ray data-‐collecLon staLsLcs. Values in parentheses are for the highest resoluLon bin.
R-‐merge = Σhkl Σi │Ii (hkl) – (I(hkl))│/ ΣhklΣi Ii (hkl) , where I i(hkl) is the ith observaLon of reflecLon hkl and (I(hkl)) is the weighted average intensity for all observaLons i of reflecLon hkl.
SET-‐UP
-‐ Gel preparaLon: agarose and HG2.6 -‐ Protein soaking (1 week/20oC) -‐ Remove protein excess and
concentraLon measurement -‐ Precipitant diffusion (20oC)
2.2. Counter difussion in gel: two layers (2L) configura1on 2.1. Preliminary crystalliza1on assays
Fig. 7. Glucose Isomerase diffrac5on results. A) Glucose Isomerase diffracLon pa|ern. B) Plot of I/σ against resoluLon to compare glucose isomerase crystal quality grown in agarosed and in HG2.6. In this case the purpose is not the comparison since they belong to different space groups. The table resumes X-‐Ray data-‐collecLon staLsLcs. Values in parentheses are for the highest resoluLon bin.
Fig. 8. TodT diffrac5on results. A) TodT diffracLon pa|ern. B) Plot of I/σ against resoluLon of TodT crystal quality grown in HG2.6. The table resumes X-‐Ray data-‐collecLon staLsLcs. Values in parentheses are for the highest resoluLon bin.
A A A
R-‐merge = Σhkl Σi │Ii (hkl) – (I(hkl))│/ ΣhklΣi Ii (hkl) , where I i(hkl) is the ith observaLon of reflecLon hkl and (I(hkl)) is the weighted average intensity for all observaLons i of reflecLon hkl.
R-‐merge = Σhkl Σi │Ii (hkl) – (I(hkl))│/ ΣhklΣi Ii (hkl) , where I i(hkl) is the ith observaLon of reflecLon hkl and (I(hkl)) is the weighted average intensity for all observaLons i of reflecLon hkl.
Fig. 1. A) RepresentaLve picture of agarose gel and HG2.6 in eppendorf. B) 2L (two layers) schemaLc configuraLon of counterdiffusion set-‐up [7].
Lzm_Agarose_NoCryo Lzm_Agarose_Cryo Lzm_HG2.6_NoCryo Lzm_HG2.6_Cryo Beamline ID23-‐1, ESRF ID23-‐1, ESRF ID23-‐1, ESRF ID23-‐1, ESRF Space group P43212 P43212 P43212 P43212 Unit cell parameters (Å) a=79,b=79, c=37.29 a=78.69,b=78.69, c=36.97 a=78.74,b=78.74, c=37.33 a=78.59, b=78.59, c=36.99 Wavelength (Å) 0.972 0.972 0.972 0.972 ResoluLon (Å) 39.50–1.20 (1.22-‐1.20) 39.35-‐1.20 (1.22-‐1.20) 39.37-‐1.20 (1.22-‐1.20) 39.29–1.20 (1.22-‐1.20) Total number of reflecLons 457266 (22508) 462926 (22039) 469290 (22705) 453679 (22146) Total unique reflecLons 37449 (1794) 36915 (1788) 37314 (1810) 36835 (1784) R-‐merge * (%) 3.6 (39.8) 6.0 (51.1) 3.9 (30.8) 3.7 (23.4) I/σ(I) 31.1 (6.2) 17.3 (3.7) 33.3 (7.8) 34.0 (9.3) Completeness (%) 99.9 (99.5) 100.0 (100.0) 100.0 (100.0) 100.0 (100.0) Redundancy 12.2 (12.5) 12.5 (12.3) 12.6 (12.5) 12.3 (12.4) B-‐factor (Å2) 11.3 10.6 11.3 11.5 Mosaicity 0.10 0.18 0.06 0.11
Glucose Isomerase_Agarose_Cryo Glucose isomerase_HG2.6_Cryo Beamline BM14U, ESRF BM14U, ESRF Space group P222 I222 Unit cell parameters (Å) a=78.19, b= 97.38, c= 128.9 a=92.33, b=98.21, c=101.7 Wavelength (Å) 0.954 0.954 ResoluLon range (Å) 44.30-‐2.56 (2.67-‐2.56) 40.57–2.56 (2.67-‐2.56) Total number of reflecLons 119120 (14433) 56389 (6839) Total unique reflecLons 31389 (3826) 14012 (1719) R-‐merge * (%) 23.8 (68.7) 11.5 (31.1) I/σ(I) 5.2 (1.7) 11.3 (4.2) Completeness (%) 97.4 (98.7) 92.6 (94.7) Redundancy 3.8 (3.8) 4.0 (4.0) B-‐factor (Å2) 7.7 4.0 Mosaicity 0.13 0.11
TodT_HG2.6_Cryo Beamline ID23-‐1, ESRF Space group P41212 Unit cell parameters (Å) a=76.66, b= 76.66, c= 36.65 Wavelength (Å) 0.972 ResoluLon range (Å) 38.33-‐1.49 (1.52-‐1.49) Total number of reflecLons 146544 (7111) Total unique reflecLons 18420 (898) R-‐merge * (%) 4.9 (37.6) I/σ(I) 22.9 (4.4) Completeness (%) 100.0 (100.0) Redundancy 8.0 (7.9) B-‐factor (Å2) 9.7 Mosaicity 0.19
B
5 4.5 4 3.5 3 2.5
5
10
15
20
2
Resolution
I/σ
I/Sigma Glucose Isomerase HG2.6 CryoI/Sigma Glucose Isomerase Agarose Cryo
B
5 4 3 2 1
20
40
60
80
2
Resolution
I/σ
I/Sigma Lys HG2.6 CryoI/Sigma Lys HG2.6 NoCryoI/Sigma Lys Agarose CryoI/Sigma Lys Agarose No Cryo
B
5 4 3 2
20
40
60
80
2
Resolution
I/σ
I/Sigma TodT HG2.6 Cryo
