Article pubs.acs.org/crystal
Cite This: Cryst. Growth Des. XXXX, XXX, XXX−XXX
Hydrogel Composite Membranes Incorporating Iron Oxide Nanoparticles as Topographical Designers for Controlled Heteronucleation of Proteins Shabnam Majidi Salehi,†,‡ Ana C. Manjua,§ Benny D. Belviso,∥ Carla A. M. Portugal,§ Isabel M. Coelhoso,§ Valentina Mirabelli,∥ Enrica Fontananova,† Rocco Caliandro,∥ Joaõ G. Crespo,§ Efrem Curcio,‡ and Gianluca Di Profio*,† †
National Research Council of Italy (CNR), Institute on Membrane Technology (ITM), Via P. Bucci Cubo 17/C, 87036, Rende, Cosenza, Italy ‡ Department of Environmental and Chemical Engineering (DIATIC), University of Calabria (UNICAL), Via P. BucciCubo 45/C, 87036, Rende, Cosenza, Italy § LAQV-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal ∥ National Research Council of Italy (CNR), Institute of Crystallography (IC), Via G. Amendola 122/O, 70126, Bari, Italy S Supporting Information *
ABSTRACT: In this study, we exploited the possibility of tuning physical−chemical properties of hydrogel composite membranes (HCMs) surfaces, by using iron oxide nanoparticles (NPs) as topographical designers, with the aim of examining the effect of surface topography and wettability on the heterogeneous nucleation of protein crystals. On the basis of roughness and contact angle measurements, it was found that surface structural characteristics, in addition to chemical interactions between the surface and protein molecules, have influence on the heterogeneous nucleation of lysozyme and thermolysin crystals to different extents. We demonstrated that increasing the amount of NPs incorporated in the hydrogel matrix promotes protein nucleation to a higher extent, potentially due to the increase of local solute concentration, arising from the enhanced wetting tendency in the Wenzel regime, and physical confinement at rougher hydrophilic surfaces. An extensive crystallographic analysis suggested the tendency of the growing crystals to incorporate hydrogel materials, which allows inducement of protein conformational states slightly different from those covered by standard crystallization methods. Protein flexibility can be thus sampled by changing the amount of NPs in the HCMs, with negligible influence on the quantity and quality of X-ray diffraction data.
1. INTRODUCTION
concentration of solute molecules is established, and this lowers the energy barrier for nucleation.6,7 Several solid materials, with a wide distribution of pore sizes and shapes, have been reported to be successful templates for crystallizing different proteins, by confining and concentrating macromolecules in the porous structure, and thereby encouraging them to form crystalline nuclei.8−10 In addition to the rigid materials with nanoscopic pores, different polymeric films also have been tested as heteronucleants for proteins. Studies on polymer surfaces with designed porosity and tailored chemical surface functionalities demonstrated the reduction in the induction time and the increase in crystal growth rate with the amount of interactive
The increasing interest to develop heterogeneous templates able to promote and control protein nucleation (heteronucleants) is currently driven by the necessity to generate welldiffracting crystals, suitable for three-dimensional (3D) structure determination at atomic resolution level by X-ray crystallography.1 Therefore, heterogeneous nucleation of proteins has been extensively investigated over the last few decades, aiming to understand the physical−chemical relationships occurring between heteronucleants and solute molecules, which may be the key factor for enabling nucleation and governing crystallization. The search for an universal heteronucleant has stimulated several studies, focusing on the influence of surface chemistry, morphology, porosity, topography, and roughness on protein− protein interaction.2−5 The general consensus is that nucleation occurs on the surface of the solid substrate when a higher local © XXXX American Chemical Society
Received: December 18, 2017 Revised: March 29, 2018
A
DOI: 10.1021/acs.cgd.7b01760 Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
Article
(see below). All chemicals were used as received. Milli-Q water was used for all solutions. 2.1.2. Synthesis of the Iron Oxide Nanoparticles. The synthesis of the magnetite nanoparticles (Fe3O4) by chemical precipitation technique was based on the procedure described by Olle.29 The particles were produced by precipitation of iron salts in alkaline medium under nitrogen atmosphere, to prevent oxidation of the particles. Iron salts solutions were prepared with FeCl3·6H2O (94.4 g) and FeCl2·4H2O (34.4 g) in aqueous solution (100 mL of distilled water) under constant magnetic stirring (1250 rpm) for 30 min in a preheated oil bath at 80 °C. Potassium oleate was then added to ensure the coating of the particles, pending about 30 min, to ensure the dissolution of the polymer. A total of 100 mL of aqueous ammonia (25%) was gradually pumped to the mixture and stirred for 30 min. Two stabilizers were also added: Hitenol-BC (100 g) and ammonium persulfate (5 g). The solution was stirred for 30 min to allow the formation of covalent bonds between Hitenol groups and oleic acid. 2.1.3. Transmission Electronic Microscopy (TEM) Analysis. The magnetic nanoparticles (NPs) were analyzed by transmission electron microscopy (TEM), using a TEM Hitachi H8100 with a LAB6 filament, used at an acceleration tension of 200 kV. The NPs sizes were determined using the ImageJ software, whereas average particle size was elicited by statistical analysis of 211 counts, using a confidence level of 95%. This analysis show an average size of 7.403 ± 0.154 nm. 2.1.4. Preparation of Hydrogel Composite Membranes. The polymer PVA (8% w/v) were dissolved in distilled water by heating (at around 80 °C) and kept under stirring for 3 h. A specific amount of NPs was then added to PVA solution to prepare solutions from 0.25 wt % to 1 wt % (Table 1) and used as topographical designers. The
sites and the enhanced adhesion of the protein solution on the surface.11−15 More recently, the ability to influence the energetic barrier to nucleation, by modulating the physicalchemical properties of polymeric porous surfaces (membranes), has been studied.16,17 It was demonstrated that, in addition to the chemical nature of the surface, membrane porosity and roughness could positively or negatively affect nucleation energy, depending on the wettability of the surface by the aqueous crystallizing solution. Hydrophobic layers generally display reduced wetting tendency, so that increased porosity or roughness will induce larger contact angle and reduced active nucleation sites. In contrast, hydrophilic surfaces with increasing porosity or roughness show a larger wetting tendency, thus reducing the activation energy to nucleation, according to the classical nucleation theory (CNT).17,18 In previous studies, we developed a membrane-assisted crystallization tool in which a hydrophobic surface, functionalized with a hydrophilic hydrogel layer, was used as heteronucleant for protein crystallization19 and as biomimetic mineralization platform.20 The resulting hydrogel composite membranes afforded for the production of protein crystals at lower solute concentration, compared to conventional vapor diffusion technique, and with enhanced diffraction properties, because of the suppression of convection currents and reduced random collisions between molecules.21−23 Furthermore, combining engineered surfaces properties and solvent removal in vapor phase through the porous membrane, provides an additional option to generate a controlled supersaturated environment in which protein crystals can nucleate and grow in a more efficient way.24−28 Here, we exploit the possibility of tuning physical-chemical surface properties of HCMs by incorporating iron oxide (Fe3O4) nanoparticles as topographical designers, providing control of membrane surface wetting and roughness, with the aim to investigate on the effect of surface properties on proteins crystallization. Extensive crystallographic analysis at synchrotron light source helped to investigate on the tendency of the growing crystals to incorporate hydrogel materials and its influence on the quantity and quality of X-ray diffraction data.
Table 1. Hydrogel Composite Membrane Samples (PVA 8% w/v) membrane samples PVA−PEDGE PVA−PEDGE−NPs 0.25% PVA−PEDGE−NPs 0.5% PVA−PEDGE−NPs 1% PVA−GA PVA−GA−NPs 0.25% PVA−GA−NPs 0.5% PVA−GA−NPs 1%
cross-linker PEDGE 3 wt PEDGE 3 wt PEDGE 3 wt PEDGE 3 wt GA 3 wt % GA 3 wt % GA 3 wt % GA 3 wt %
nanoparticles % % % %
Fe3O4, 0.25 wt % Fe3O4, 0.5 wt % Fe3O4, 1 wt % Fe3O4, 0.25 wt % Fe3O4, 0.5 wt % Fe3O4, 1 wt %
embedded NPs allow for the modulation of surface topography by adjustment of the NPs content and by magnetic manipulation based on the magnetic susceptibility of these particles. The study included in the present paper reports only the effect of NPs load in the surface topography. To obtain good dispersion of nanoparticles, ultrasonication was employed for 30 min immediately prior to the crosslinking reaction. Then, the 3 wt % cross-linker (PEDGE or GA), and hydrochloric acid 0.1 M, used as catalyst, were added, respectively, to complete the prepolymerization solution. Then hydrogel solution was cast on the PP membrane using a film applicator (Electrometer 4340, automatic film applicator) adjusted at 50 μm thickness. To facilitate the coating process, the hydrophobic PP support was conditioned by soaking in methanol overnight to increase the adhesion with the hydrophilic coating layer. The cross-linking reaction was completed in almost 3 h. PVA hydrogel composite membranes without NPs were prepared with both GA and PEDGE as cross-linker and used as reference samples. 2.2. Characterization of Hydrogel Composite Membranes. 2.2.1. Optical Microscopy. PVA hydrogel composite membranes with randomly dispersed NPs were observed using an optical microscope (DM 2500M, Leica Microsystems, Germany) equipped with a video camera. 2.2.2. Scanning Electron Microscopy (FEG-SEM-EDS). The samples were coated with a Cr film of 20 nm thickness, using a sputter coater from Quorum Technologies (model Q150TES) and analyzed in a FEG-SEM system from JEOL (model JSM7001F) equipped with a
2. EXPERIMENTAL SECTION 2.1. Materials and Methods. 2.1.1. Chemicals. Polypropylene (PP) flat sheet membranes (Accurel PP 2EHF, nominal pore size 200 nm, overall porosity 70%) from Membrana GmbH (Wuppertal, Germany) were used as support for HCMs preparation. Lysozyme from chicken egg white (HEWL, Mw 14307 Da, pI 11.35, cod. 62970), Thermolysin from Bacillus thermoproteolyticus (Mw 37500, pI 4.45, cod. T0331), poly(vinyl alcohol) (PVA, average Mw 150 000 g/mol, 98.9% hydrolyzed, cod. 101302902), glutaraldehyde (GA, grade II, 25% in H2O, cod. MKBG3597V), poly(ethylene glycol) diglycidyl ether (PEDGE, average Mw 500 g/mol, cod. MKBL8500V), ferric chloride hexahydrate (97% FeCl3·6H2O, cod. 207926), ferrous chloride tetra hydrate (99% FeCl2·4H2O, cod. 380024), potassium oleate (40 wt % paste in water, CH3(CH2)7CHCH(CH2)7COOK, cod. 60420), ammonium persulfate (>98% (NH4)2S2O8, cod. 431532), hydrochloric acid (HCl, 37%, AR grade, cod. 433160), sodium chloride (NaCl, cod. 71376), sodium acetate (CH3COONa, cod. 71183), 2-(N-morpholino)ethanesulfonic acid hydrate (MES, cod. 69890), dimethyl sulfoxide (DMSO, cod. 41639), and ammonium sulfate ((NH4)2SO4, cod. 09978) were bought from Sigma-Aldrich. Ammonium hydroxide (NH4OH, cod. 1336216) was from Mallinckrodt, and Hitenol-BC was from Daiichi KogyoSeiyaki. Methanol (CH3OH, HPLC grade, cod. 20844.320) was from VWR. Iron oxide nanoparticles were prepared by a chemical coprecipitation technique B
DOI: 10.1021/acs.cgd.7b01760 Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
Article
programs and of the program RootProf,37 to calculate and plot group averages of the parameter values. 2.5. Structural Analysis. The set of 64 new lysozyme crystal structures obtained by the crystallographic analysis were compared residue-by-residue by using Cartesian coordinates of their Cα atoms and backbone dihedral angles. The former was used to calculate the root-mean-square deviation (RMSD) between each pair of structures, though the program Superpose.38 The latter was used to calculate the protein angular dispersion (PAD) of the ensemble of structures through the script TPAD39 run on VMD.40 PAD measures the spread of the backbone dihedral angles among the different crystal structures and quantifies the residue-by-residue flexibility. It proved successfully in identifying anomalous protein plasticity upon pathogenic mutations41,42 or ligand binding43 in molecular dynamics simulations.
coupled EDS detector with light energies from Oxford (model INCA 250). 2.2.3. Contact Angle Measurements. The values of the contact angle of water on HCMs were measured using CAM 101-Optical contact angle and surface tension meter (KSV Instruments Ltd., Helsinki, Finland), at room temperature. A small drop (2 μL) of the solution was placed on the sample surface by a microsyringe, and measurements were carried immediately. The contact angle values reported in the following are the average of five consecutive measurements for each sample. 2.2.4. Profilometry Measurements. The analysis of surface roughness of the PVA hydrogel coating layers was initially attempted through atomic force microscopy (AFM). However, the topographical features presented in hydrogel layers with a higher content of magnetic particles were within the micrometer scale, at the limit of the AFM analytical range size, not allowing eliciting information on the influence of NPs on surface roughness in this case. For this reason, surface roughness profiles were measured by a DEKTAK profilometer, using a 5 μm radius tip with 1 mg stylus load and at the velocity of 0.1 mm/s. Profilometry was used instead of AFM because there is no limitation for sticking the tips to the surface of the hydrogel. The scanner sizes were 0.5 × 0.5 mm2. The average roughness, defined as the arithmetic average of the absolute values of the surface height deviation measured from the mean plane surface, and the root-meansquare (rms) roughness, defined as the standard deviation of the surface profile from the mean plane surface, were calculated by Nanoscope software. The average value of three measurements per sample is reported. 2.3. Protein Crystallization on HCMs. Lysozyme solution, at the initial concentration of 40 mg/mL, was prepared in 0.1 M sodium acetate buffer at pH 4.6 and mixed 1:1 volume ratio (5 + 5 μL) with the reservoir solution made of 7% w/v sodium chloride in 0.1 M sodium acetate buffer at pH 4.6. For thermolysin crystallization tests, protein solution at the initial concentration of 77 mg/mL was prepared in 50 mM DMSO 45% and MES buffer at pH 6.2 and a mixed 1:1 volume ratio (5 + 5 μL) with 1 M sodium chloride prepared in the same buffer. In this case, reservoir solution was made by 1 M ammonium sulfate. Crystallization tests were carried out by using 24well plates (from Qiagen) conventionally used for the vapor diffusion technique and adapted for membrane-assisted crystallization experiments (sitting drop technique). The setup consisted of a 10 μL drop (equal volumes of protein and reservoir solutions) placed on a HCM disc (1 cm diameter) fixed on a glass cover-slide, and 500 μL of precipitant solutions as reservoir. The crystallization tests were carried out at 20 °C with five replications for each condition to test the reproductively of the results. Crystallization trials were also carried out by using the untreated PP membrane or the conventional glass coverslips as templates, for reference. Crystal growth was monitored at regular times through a stereomicroscope (Nokia AZ100) equipped with a video camera. 2.4. Crystallographic Analysis. Diffraction properties of protein crystals were checked by using the X-ray beam generated at the Diamond Light Source of Oxford (UK), beamline I04. Data collections were carried out in cryogenic conditions (100 K), under beam energies of 1268 eV. The XDS program30 was used to perform data reduction, while POINTLESS and AIMLESS programs31 were used to find the space group symmetry and to scale the diffraction data. The structures were solved by molecular replacement (MR), with the REMO program32 included in the package ILMILIONE,33 by using the crystal structures 4N9R34 and 5MNR35 as MR models for lysozyme and thermolysin, respectively. The matching between experimental and calculated electron densities was improved by performing an automatic building procedure on the structure obtained with MR in “rebuilt-in-place” mode, by using Autobuild wizard36 included in PHENIX.36 A total of 99 crystals were analyzed at synchrotron, resulting in 68 crystal structure determinations (52 for lysozyme, 16 for thermolysin). The statistical analysis of crystallographic parameters was performed by means of python scripts to collect parameter values from output files of indexing and phasing
3. RESULTS AND DISCUSSION 3.1. Hydrogel Composite Membranes Preparation. A layer of PVA hydrogel, with and without Fe3O4 nanoparticles, was cross-linked with GA or PEDGE on the porous surface of the PP support, under acidic conditions. Representative optical microscopic image of a PVA−PEDGE composite membrane (Figure 1), embedded with NPs at concentration of 1 wt %,
Figure 1. Surface of a HCM constituted by PVA hydrogel layer, crosslinked with PEDGE and embedded iron oxide NPs (1 wt %).
shows the dispersion of iron oxide particles or particle clusters (with size within the micrometer scale range) through the PVA hydrogel layer, which still forms despite the coating of NPs with potassium oleate. Particle clusters should mainly be formed before and/or along the cross-linking reaction, since polymer cross-linking should hamper the mobility of NPs and the formation of aggregates. A typical FEG-SEM image of the surface and the cryofractured cross-section of a HCM sample is shown in Figure 2. It displays the uniform and defect free thin hydrogel layer (∼10 μm thick) on the surface of the PP porous membrane (∼170
Figure 2. Characteristic SEM image of the top (left) and cross-section (right) of PVA−GA−NPs 1% supported on porous PP (see Table 1). The inset of the figure on the left side displays the EDS analysis performed on the surface of the HCM. C
DOI: 10.1021/acs.cgd.7b01760 Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
Article
Figure 3. Three-dimensional profilometry images of HCMs surfaces: (A) PVA−PEDGE, (B) PVA−PEDGE−NPs 0.25%, (C) PVA−PEDGE−NPs 1%, (D) PVA−GA, (E) PVA−GA−NPs 0.25% , (F) PVA−GA−NPs 1%.
μm thick). The cross-section image reveals that the hydrogel phase does not penetrate thoroughly the porous PP, due to the hydrophobic nature of the support, that prevents the aqueous prepolymerization solution to penetrate in the porous structure. Iron oxide particles were detected by EDS on the surface (inset of Figure 2) as well as throughout the hydrogel layer. Analysis of SEM-EDS also reveals the presence of Cr, which is due to the samples precoating needed for analysis. Three-dimensional profilometry images of HCMs, obtained with a scanner size of 0.5 × 0.5 mm2, are shown in Figure 3. Figure 3A,D displays the relative smooth topography of HCMs prepared without NPs. The presence of NPs leads to the formation of eddies on the hydrogel layer (Figure 3B,C,E,F), which results in larger values of roughness as the load of NPs increases, as evidenced by both average (Ra) and root-meansquare (RMS) roughness values reported in Table 2. The water contact angles (CA) for all PVA hydrogel composite membranes range from 43° to 56°, which is much lower than the CA of the pristine hydrophobic PP substrate
Table 2. Average Roughness (Ra), Root-Mean-Square Roughness (RMS) and Contact Angle (CA) of the HCMs HCM samples PVA−PEDGE PVA−PEDGE−NPs 0.25% PVA−PEDGE−NPs 1% PVA−GA PVA−GA−NPs 0.25% PVA−GA−NPs 1%
Ra (nm) 301 562 811 361 628 821
± ± ± ± ± ±
RMS (nm)
25 63 145 69 111 137
370 719 1018 448 848 1036
± ± ± ± ± ±
20 40 164 90 164 172
CA (deg) 49 45 43 56 50 48
± ± ± ± ± ±
2 3 2 1 2 2
(137°) (Table 2). These values confirm the increase in hydrophilicity due to the PVA component. PVA hydrogel crosslinked with PEDGE exhibits a slightly larger hydrophilic character than PVA−GA samples. Contact angle measurements reveal also an additional moderate rise in hydrophilicity upon incorporation of an increasing load of iron oxide. On the bases of the Wenzel theory,44 hydrophilic surfaces (CA < 90°) with a typical size of roughness details smaller than D
DOI: 10.1021/acs.cgd.7b01760 Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
Article
the size of the interacting droplet and without air pockets (referred to as a homogeneous interface with complete wetting), become more hydrophilic with the increase in roughness. According to data reported in Table 2, the enhanced hydrophilic character of the HCMs with the increasing amount of NPs can be therefore associated with the combined effect of the higher water affinity of the iron oxide nanoparticles themselves, and the increase in the surface roughness. When a droplet of protein solution is placed on the HCMs surfaces, a different spreading pattern can be observed, depending on the surface wettability and roughness.45 Surface topography or fractal structure of hydrophilic HCMs can provide more areas to accommodate the adsorbed protein molecules under a Wenzel-type wetting regime (Figure 4). In
Figure 5. Optical microscopy images of lysozyme crystals obtained with HCMs after the same observation time (4 days): (A) PVA− PEDGE; (B) PVA−PEDGE−NPs 0.25%; (C) PVA−PEDGE−NPs 1%; (D) PVA−GA; (E) PVA−GA−NPs 0.25%, and (F) PVA−GA− NPs 1%.
presumably identical, batches of solution are subjected to the crystallization protocol simultaneously. The probability of nucleation is estimated by considering the fraction of droplets that contained a crystal after 4 days (the time over which crystallization was considered completed) for every HCM sample (Figure 6A). Each droplet behaves as a single batch system and a large number of such systems permits deriving the required statistics. The average number density corresponds to the mean number of crystals obtained for each condition, considering five replicates (Figure 6B). The results shown in Figure 6 clearly demonstrate the enhancement in the probability of nucleation and in the density number by increasing the loading of NPs, as already anticipated by microscopic observations. Results are consistent with a probability of nucleation that is almost 100% when incorporating the NPs in the hydrogel layer, and the density numbers increase by about four times when using NPs at 1 wt %, compared to HCMs without nanoparticles in the hydrogel matrix. According to our previous studies,24 protein crystallization by means of HCMs occurs by two sequential adsorption modes, operating at low and higher protein concentration, in agreement with the Langmuir−Freundlich equilibrium isotherms model.46 Initially, at low concentration, macromolecules spread over the surface of the hydrogel layer by noncooperative random adsorption, until the first monolayer is established. Then, as the solution concentrates by solvent removal in the vapor phase, the transition from noncooperative to cooperative adsorption occurs at a certain solute concentration, so that a second layer grows onto the first. The nonspecific interaction between proteins and PVA polymer network enables macromolecular adsorption by the hydrogel layer, thereby concentrating molecules, while reducing, to some extent, molecular mobility. In fact, lysozyme, with isoelectric point 11.35, is positively charged at the working pH 4.6, while thermolysin, having pI 4.45, is negatively charged at pH 6.2. Accordingly, both charged proteins display some interacting affinity for the network with higher density of polar groups (un-cross-linked −OH groups of PVA). Furthermore, because the Hitenol-BC contains a sulfonate group that confers a negative charge to the particles,29 positively charged lysozyme is expected to interact at higher extent with the membrane surface than thermolysin. In the case of HCMs without NPs, the smooth topography of the hydrogel layer allows macromolecules adsorbed on the surface to stay apart from each other (Figure 7 left), so that, in
Figure 4. Protein solution droplet (A) on a smooth surface and (B) on the rough surface of HCMs embedded with NPs. The amount of adsorbed molecules on a fragment with a fractal surface significantly exceeds that on one with flat surfaces in a Wenzel-type wetting regime.
such conditions, from the classical theory, the free energy barrier for heterogeneous nucleation is found to decrease with the contact angle and surface roughness.15,16 Furthermore, the presence of local topographical niches induced by the hillocks generated by the NPs dispersed at the hydrogel surface enhances the number of sites where a higher local concentration of protein molecules is established by physical entrapment. Since different surface features lead to a diverse heterogeneous crystallization behavior, the distinct topographies obtained in this work by dosing the amount of dispersed NPs in the hydrogel layer were expected to lead to variant energy barriers for heterogeneous nucleation. 3.2. Proteins Crystallization Studies. Extensive screening tests were performed by using well plates, adapted to work with composite membranes, aiming to investigate on the influence of HCMs features and NPs loading on the crystallization of two model proteins. When HCMs without nanoparticles are used as substrates for lysozyme crystallization, mostly no crystals or only a few crystals were observed over a fixed range of time (Figure 5A,D). For the HCMs with embedded NPs, a number of crystals are obtained for the same observation time, and this number increases with the amount of NPs introduced in the hydrogel matrix, for both PVA−GA and PVA−PEDGE composites (Figure 5B,C,E,F). A further observation on images of Figure 5, corroborated with similar observations on other images, is that crystals grown on PVA−GA−NPs typically have rounded edges, contrary to those grown with PVA−PEDGE, which have sharp edges. This would be indicative of a potential larger inclusion of hydrogel material within the growing crystal in the PVA−GA−NPs case. To show the influence of HCMs properties on lysozyme crystallization quantitatively, the probability of the nucleation event and the density number of crystals have been calculated for the several conditions. The experimental determination of the probability of nucleation is approximated by the fraction of droplets where crystallization occurred, in which many, E
DOI: 10.1021/acs.cgd.7b01760 Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
Article
Figure 6. (A) Probability of nucleation and (B) average density number of the lysozyme crystals observed at the increasing amount of NPs in HCMs. The number of batches that produce at least one crystal, N*, is monitored with time, and the nucleation probability is obtained as F(t) = N*(t)/N, where N is the total number of batches.
PVA−GA and ∼23.3 for PVA−PEDGE HCMs. Therefore, the difference in molar weight between the two cross-linkers allows a 5 times higher amount of un-cross-linked −OH groups to exist in the PVA−PEDGE HCMs. Crystal growth rates for lysozyme and thermolysin are shown in Figure 8. They have been estimated by calculating the first
Figure 7. Schematic illustrating the effect of molecular confinement on proteins aggregation. Blue lines denote the hydrogel mesh structure with polymer chains and cross-linking points. On the left: smooth hydrogel surface with no hillocks, where protein molecules are initially adsorbed on the flat substrate and kept apart from each other; on the right: molecules interacting on an irregular surface topography where physical confinement enhances contact points among them. The polymer mesh structure drawn here does not necessarily represent the effective system, but it is intended to illustrate the role of confinement effects. Macromolecules are indicated with one end colored in dark blue, which interacts preferentially with the polymer chain, and the other end colored lighter, which is involved in self-interactions. The size of the solute relative to the mesh size is drawn not to scale.
combination with reduced molecular mobility effects, protein− protein interaction is less favored. When heterogeneous nucleation takes place on rough hydrophilic surfaces, the hillocks provide larger area to accommodate the adsorbed protein. Furthermore, the physical entrapping of molecules in the concaves results in higher local protein concentration, which increases the possibility of nucleation, compared to a flatter surface (Figure 7 right). In addition, the hillocks characteristic of the rough surfaces may pack molecules in different ways, and the resulting diversity in the bond angle for the protein molecules with its neighbors increases the probability of aggregation.16 The enhanced nucleation rate observed for NPs-loaded HCMs is therefore due to the increased number of available locations where energetic barrier to nucleation is overcome, corresponding to irregularities of the surface, generated by the increasing load on NPs. Notably, of the two cross-linkers used in this work, PEDGE-based composites display a higher aptitude to stimulate lysozyme nucleation than PVA−GA samples. This might be due to the larger density of un-cross-linked polar groups existing in the mesh structure of the hydrogel in the former case that increases the number of interaction sites. In fact, considering the weight amounts of the two components, the molar ratio between the −OH groups in the monomer and the cross-linker is ∼4.7 for
Figure 8. Crystal growth rate measured at different times for lysozyme (a) and thermolysin (b) crystallized on PVA−GA (GA) and PVA− PEDGE (PEG) HCMs with different amount of NPs. Only cases where the protein crystal could be clearly seen at the microscope were included in the histograms. Only positive error bars are shown, calculated by propagating the instrumental error.
derivative of the measured crystal size with respect of the observation time. Lysozyme is characterized by a faster growth rate with respect to thermolysin, for which no crystals were observed before 2 days. For lysozyme, the effect of HCM-NPs is to enhance the crystal growth, which occurs earlier than in the glass or PP case. Growth rates higher than 50 μm/h are reached by using HCMs, which become around 100 μm/h in the presence of NPs. The same effect is not observed for thermolysin, whose crystal growth rates are around 15 μm/h independently of the support used. Furthermore, in the case of F
DOI: 10.1021/acs.cgd.7b01760 Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
Article
Figure 9. Crystallographic parameters for lysozyme measured from 6 crystals grown in standard sitting drop (glass), 7 crystals grown on PP supports, 24 crystals grown in PVA−GA HCMs, and 30 crystals grown in PVA−PEDGE HCMs. Error bars represent the standard deviation of the parameter in each bin. They are zero in the case of single entry. Parameter Res is the data resolution (in Å); thermal factor is the overall thermal parameter (in Å2), as determined by the data normalization procedure; Rmerge is the crystallographic agreement factor between amplitudes of symmetrically equivalent reflections; mosaicity is a measure of the spread of crystal plane orientations, estimated from the diffraction spot profiles.
Figure 10. Structural model parameters determined from lysozyme data sets that converged to crystal structure solution, derived from 6 crystals grown in standard sitting drop (glass), 6 crystals grown on PP supports, 15 crystals grown in PVA−GA HCM,s and 25 crystals grown in PVA− PEDGE HCMs. Error bars represent the standard deviation of the parameter in each bin. They are zero in the case of single entry. Parameters a and c are the length (in Å) of the crystal cell axes; Solvent is the fraction of water contained in the crystal; Rfree is the crystallographic agreement factor between amplitudes observed and calculated from the structural model on a subset of reflections not used during the structural refinement procedure.
G
DOI: 10.1021/acs.cgd.7b01760 Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
Article
HCMs with NPs, and therefore no inclusion of Fe particles from the hydrogel to crystals occurred. The structural models obtained from lysozyme crystals can be compared by analyzing the matrices shown in Figure 11,
lysozyme, with PVA−GA−NPs a faster crystal growth than PVA−PEDGE−NPs HCMs was detected. This observation might suggest a larger inclusion of hydrogel material in the growing crystals for PVA−GA−NPs. In fact, it is known that a growing crystal exerts pressure on its surroundings, which depends on the supersaturation.47 When interconnected polymer networks, such as hydrogels, are encountered during crystal growth, the gel network can respond to the crystallization pressure by readjusting.48 The ability to rearrange is related to the modulus of the gel network and the rate of crystal growth.49 Once the gel network is strong enough to sustain this pressure, the crystal is forced to grow around the gel fibers and hence incorporate them.50−52 Otherwise, if the gel is weak, the growing crystals damages the network, and it is pushed away, leading to exclusion of the polymer.48,49 It has been also demonstrated that some proteins52 could, otherwise, incorporate gels even though the magnitude of the crystallization pressure is much higher than the gel strength under the effect of the high growth rate. Here, it is argued that the higher growth rate observed for lysozyme crystals grown when using PVA−GA HCMs would have increased the entrapping of hydrogel fibers in the crystal structure, as already found in previous studies on calcite,53,54 calcium tartrate tetrahydrate and glycine,55 and some protein24,56,57 crystals grown in gel media. 3.3. High-Throughput Crystallographic Analysis. Aiming to investigate on the effect of potential gel inclusion in the growing crystals in the several explored conditions, a highthroughput crystallographic analysis has been performed at the synchrotron light source. The statistical analysis of the crystallographic parameters of lysozyme crystals is shown in Figure 9. The B factor, describing the overall thermal motion of protein atoms, is increased by using HCMs. Other parameters describing the diffraction data quality (Rmerge) and quantity (Res), and crystal quality (mosaicity) are not influenced by the presence of HCMs or NPs in the case of PVA−PEDGE. Instead, the presence of PVA−GA HCMs worsen the properties of the diffraction pattern, and this occurs especially for NPs at 1 wt %. These results are in agreement with previous observations, showing that the hydrogel interferes with crystal growth, entering in the crystal habit, hence deteriorating its properties.28 The enhanced growth rate observed for PVA−GA compared to PVA−PEDGE HCMs, and the consequent larger incorporation of hydrogel material in the crystals, explains this effect. For thermolysin crystals, no clear trends in the crystallographic parameters can be outlined, due to limited statistics (see Figure S1). Parameters related to the structural models obtained by the lysozyme diffraction patterns, plotted in Figure 10, evidence that HCMs induce slight distortions of the crystal cell, namely, a decrease of the a parameter and an increase of the c parameter, which increase with the NPs content, for NPs < 1 wt %. As a consequence, the solvent content (Solvent) is slightly decreased in the same conditions. The cell distortions occur even at NPs 1 wt % for PVA−GA HCMs. A similar distortion is observed for the a parameter of the thermolysin crystal cell, even in this case the overall change of the solvent content is negligible (Figure S2). Notably, the parameter that describes the quality of both lysozyme and thermolysin structural models (Rfree) is not affected by the use of HCMs or NPs at different concentrations. It should also be noticed that no heavy atom was found in structural models derived from crystals grown on
Figure 11. Matrix of root mean square deviations (RMSD) calculated between pairs of structural models refined from lysozyme crystals grown on PVA−GA (top) and PVA−PEDGE (bottom) HCMs.
collecting the pairwise RMSD values, which measure deviations in the position of the Cα atoms between superposed structures. Very small average deviations are generally introduced by HCMs, which are
