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Langmuir 1998, 14, 5568-5572
Self-Assembly of Polypeptide Molecules on Charged Surfaces. 1. Effect of Polydispersity Vladimir Kitaev and Eugenia Kumacheva* Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6 Received April 21, 1998. In Final Form: July 8, 1998 Atomic force microscopy and dynamic light scattering were used to study the effect of polydispersity on surface-generated fiber formation of poly[(γ)-benzyl-L-glutamate] (PBLG) from solutions in dioxane. Polydispersity was modeled by mixing at varying weight ratios of PBLG samples with essentially different molecular weights. We found three distinctive regimes of fibrogenesis of PBLG determined by the ratio of shorter-to-longer polymer in the solution. At equal weight concentrations of longer and shorter molecules, the shorter molecules irreversibly attach to the surface, while the longer polymer assembles on the top of the primary layer. A reduction of the weight ratio to ca. 1:20, produces large, distorted fibers consisting of a mixture of long and short polymers. A further decrease of the ratio shorter-to-longer macrochains to the value as small as 1:200 induces lateral association of the surface-attached fibers formed mostly from a longer polymer.
Introduction Synthetic polypeptides are useful models for studies of the fibrogenesis of molecules with essential rigidity. A characteristic feature of polypeptide fibers self-assembled from solutions in organic solvents on various surfaces is that they consist of aggregates or clusters of molecules.1-3 Similar intermediate structural units have been observed in fibers formed by association of proteins in aqueous solutions.4 In recent studies of the self-assembly of poly[(γ)-benzylL-glutamate] (PBLG) on a mica surface, we observed a surprising monodispersity of these intermediate clusters. In fibers formed by PBLG with the molecular weight 300 000 from solutions in dioxane, the length and width of clusters were 140 ( 10 and 35 ( 10 nm, respectively. Similar features were observed in PBLG clusters selfassembled from solutions in dimethylformamide. Such monodispersity implies that the dimensions of clusters determined by the number of molecules constituting them are controlled by thermodynamic factors. Particularly, it was suggested that the number of molecules in a cluster is determined by a collective interaction of the molecules required to bring them to an unfolded configuration.1 We sought to see the growth of clusters as a surfacenucleated process. The attachment of the first individual polypeptide molecules to the substrate was driven by the electrostatic interaction between the dipoles of PBLG and charged mica surface. Further cluster growth occurred via an antiparallel side-to-side intermolecular association of the polypeptide molecules by analogy with the association of sufficiently long chains in concentrated solutions.5,11 In the suggested scheme, a number of questions remain open. What happens if the associating molecules are not complementary, e.g., have different shape or length? What would be the result of the distorted side-to-side association when a small fraction of molecules that associate in a (1) Kitaev, V.; Schille´n, K.; Kumacheva, E. J. Polym. Sci.: Polym. Phys. 1998, 36, 1567. (2) Aritake, T.; Tsujita, Y.; Uematsu, I. Polym. J. 1975, 7, 21. (3) Masuda, Y.; Osumi, M. J. Electron. Microsc. 1974, 23, 303. (4) Trelstad, R. L.; Hayashi, K.; Gross, J. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 4027. Gelman, R. A.; Williams, B. R.; Piez, K. A. J. Biol. Chem. 1979, 254, 180. (5) Pyzuk, W.; Krupkowski, T. Macromol. Chem. 1977, 178, 817.
different way is present in the solution? For example, PBLG molecules with a relatively low molecular weight tend to associate end-to-head and give a mixed (end-tohead and antiparallel) association when the molecular weight is increased.5 A more general question is how sensitive are well-organized self-assembled fibers to the presence of foreign molecules? Recent studies of fibrogenesis of β-amyloid peptides indicate that addition of short peptides leads to inhibition of fibrillization of longer chains.6 Finally, what would be the competition in adsorption of longer and shorter macromolecules forming structures more complex than monolayers? Generally, in dilute and moderately concentrated solutions of flexible homopolymers there is a preference in adsorption of longer chains vs shorter chains due to a smaller loss of the translational entropy in comparison with the bulk solvated state.7 However, for rigid rod molecules such as PBLG, the loss of entropy is less significant than it is for random coil polymers. Moreover, when polymer adsorption is accompanied by the formation of supramolecular structures, the entropic argument is not obvious. These questions have practical relevance in addition to scientific importance. Polymer polydispersity can be a reason for the growth of large fibers or shapeless aggregates, an undesirable phenomenon in material science. For biopolymers, the association of abnormally expanded peptide repeats in proteins with the formation of β-sheets can cause inherited neurodegenerative diseases.8 The aim of this work is to investigate the effect of PBLG polydispersity on surface-induced fibrogenesis. Polydispersity was modeled via a bidisperse mixture of polymer chains that had a large difference in their molecular weights. Polymers were dissolved in organic solvents at different weight ratios, while the total polymer concentration in the solution corresponded to a dilute regime. In different series of experiments, we found three distinctive (6) (a) Blanchard, B. J.; Konopka, G.; Russel, M.; Ingram, V. M. Brain Res. 1997, 776, 40. (7) Fleer, J.; Cohen Stuart, M. A.; Scheutjens, J. M. H. M.; Cosgrove, T.; Vincent, B. Polymers at Interfaces; Chapman & Hall: London, 1993; p 502. (8) Perutz, M. F.; Johnson, T.; Suzuki, M.; Finch, J. T. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5355.
S0743-7463(98)00455-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/27/1998
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Figure 1. Various magnification AFM images of fiberlike layers self-assembled on the mica surface from 1,4-dioxane solution of (a), (b) PBLG-300K and (c), (d) PBLG-20K; cPBLG-300K ) 9.5 × 10-5 g/mL, cPBLG-20K ) 9.5 × 10-5 g/mL.
types of fibrogenesis determined by the weight ratio shorter-to-longer macromolecules in the solution. It has to be emphasized that, due to the dominant role of the substrate in surface self-assembly, the morphology of the polypeptide associates can be very different in the bulk solution and on the surface. Second, polymersurface interaction induces fiber formation at sufficiently low polymer concentration in the bulk, i.e., when no or very weak interaction of PBLG molecules occurs in the solution. Experimental Section Materials. Poly[(γ)-benzyl-L-glutamate] polymer samples were purchased from Sigma Chemical Co. and used as supplied. The higher molecular weight polymer (referred to as PBLG-300K) had the molecular weight MV ) 296 000 and MW ) 308 000 as determined by intrinsic viscosity and light scattering measurements (batch 35H5532). The lower molecular weight polymer (referred to as PBLG-20K) had MV ) 22 000 and MW ) 17 400 (batch 35H5531). 1,4-Dioxane (ACS grade) was received from ACP Chemicals Inc. and used as received. For controlled experiments, dioxane (99.8%, anhydrous) was obtained from Sigma-Aldrich and used from freshly opened bottles. All solutions were prepared 27 ( 9 h before experiments on surface self-assembly were carried out. Adsorption of polypeptides on the mica surface is described elsewhere.1 Freshly cleaved mica samples were exposed to filtered PBLG solutions for 17 ( 3 h, then washed in a pure dioxane, dried in the laminar hood for 2 h, and immediately studied with atomic force microscopy (AFM). Methods. Nanoscope III (Digital Instruments, Santa Barbara, CA) with a pyramidal Si3N4 tip was used for structural studies of PBLG layers. The spring constant was 0.58 or 0.38 N/m in experiments with self-assembled PBLG-300K and 0.06
or 0.12 N/m when mixed fibers or fibers composed of a pure PBLG20K were imaged. Scan rates varied in the range from 1 to 4 Hz. Most of the images presented in this paper were obtained in a contact mode. However, to check the possibility of a distortion of the polypeptide structure by a tip, some of the AFM measurements on PBLG-20 K were performed in a tapping mode. Each sample was examined at 3-5 different points. Dynamic light scattering (DLS) experiments were carried out using an argon-ion laser (514.5 nm/488 nm) with an adjustable output power from Lexel Laser Inc. (model Excel 300) as a light source. The angle of detection of the scattered light was varied from 40 to 135° using a Brookhaven goniometer. A Brookhaven digital correlator (model BI-2030 AT) with 136 channels controlled by a PC (LS242) was used for data collection. The DLS data analysis is described elsewhere.1 All measurements were performed at 21.8 °C.
Results and Discussion Self-Assembly of Individual Polymers. Typical AFM images of the fiberlike layers formed by individual polymers from the solutions in dioxane at polymer concentration cpol ) 9.5 × 10-5 g/mL are presented in Figure 1. Well-organized fibers of PBLG-300K with the thickness 140 ( 10 nm randomly attach to mica covering ∼70 % of the surface (Figure 1a). Repeated scans do not noticeably affect the morphology of the fiberlike layer. The structure of fibers at a higher magnification is displayed in Figure 1b. Clusters forming fibers are asymmetric and have the dimension of the long axis ∼140 ( 10 nm, i.e., close to the persistence length of PBLC molecules.9 Fibers assembled from the solution of PBLG20K in dioxane (Figure 1c) are easily distinguishable from (9) Block, H. Poly(γ-benzyl-L-glutamate) and other glutamic acid containing polymers; Gordon and Breach: New York, 1983; 215 pages.
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Figure 2. Typical images of PBLG layers adsorbed on the mica surface from mixed dioxane solutions of PBLG-300K and PBLG-20K at equimass polymer ratio. cPBLG-300K ) 3 × 10-5 g/mL, cPBLG-20K ) 3 × 10-5 g/mL. Time of the surface exposure to the solution: (a) 20 h; (b) 1 week.
those formed by PBLG-300K. First, PBLG-20K fibers have a smaller thickness (about 60-70 nm). Second, the layer formed by PBLG-20K is softer: when force exerted by the tip on a sample is not sufficiently minimized, there is a difference in structure of the layer observed in the first and successive scans (the latter is shown in the center of the image). Repeated scans increase fiber orientation and the roughness of the structure formed by PBLG-20K. This feature was observed earlier in AFM studies of the Langmuir-Blodgett films of similar “hairy-rod” polyglutamate.10 A higher compliance was used as a diagnostic to identify fibers composed from PBLG-20K or a mixture of PBLG-300K and PBLG-20K. Finally, in contrast to PBLG-300K, the whole surface of mica is uniformly covered with PBLG-20K. A higher surface coverage can be expected from the ability of PBLG-20K to associate in both antiparallel and end-to-head fashion, providing easier interaction of macrochains in the vicinity of the surface with the molecules already attached to the mica. Inspection of the fiber structure at a higher magnification (Figure 1d) reveals a reduced compactness of the clusters in fibers in comparison to the longer polymer. Self-Assembly of Mixed Polymers. As shown in Figure 2a, polymer self-assembly from the solution containing a mixture of PBLG-300K and PBLG-20K at equimass ratio (1:15 molar ratio) produces a close-packed layer of fibers of PBLG-20K in the primary layer with (10) Tsukruk, V. V.; Foster, M. D.; Reneker, D. H.; Schmidt, A.; Knoll, W. Langmuir 1993, 9, 3538.
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fibers of PBLG-300K attached to the top of it. This morphology is stable for over 1 week of exposure of the mica surface to the solution of longer and shorter macromolecules. No evidence of any change in the structure of PBLG-300K fibers in the top layer or replacement of the PBLG-20K from the bottom layer was observed (Figure 2b). The total height of the polymer assembly remains ca. 10-12 nm; i.e., it corresponds roughly to the thickness of approximately two layers of fibers formed from individual polymers. Experiments on the consecutive adsorption of PBLG molecules with different molecular weights were carried out in the following way. A mica surface was exposed to a solution of the individual polymer (PBLG-300K or PBLG20K) (cpol ) 9.5 × 10 -5 g/mL) in dioxane for the time interval needed to achieve an equilibrium adsorption (usually 12 ( 3 h), then washed in a pure solvent, and placed into a solution of the polymer with a different molecular weight for 20 h. In a different series of the experiments, longer (or shorter) macromolecules were adsorbed onto the surface from the solution of the individual polymer (cpol ) 6 × 10 -5 g/mL), and after the equilibrium adsorption was attained, a shorter (or longer) polymer was added to the solution at the same concentration (cpol ) 6 × 10-5 g/mL). Both types of experiments gave a similar result. Regardless of the molecular weight, the morphology of the top layer observed on the mica was always representative of that of the last polymer added to the solution, while the total thickness of the polymer layer corresponded to that of two layers of fibers formed by individual polymers. These observations suggest that neither longer nor shorter macromolecules replace each other from the surface of mica. At equimass ratio in the solution, molecules of PBLG-20K adsorb to the surface first due to kinetic reasons and form fibers. Adsorption is driven by the electrostatic interaction between the PBLG dipoles and charged surface. Association of longer species with the surface-attached shorter molecules occurs via a dipole-dipole interaction.5,11 This interaction leads to the formation of fibers in the second layer.12 If longer macromolecules adsorb first (experiments on consecutive adsorption), the scenario is essentially the same. The absence of replacement is caused by a collective adsorption of molecules forming fibers in the first layer. In addition, polymer-polymer interaction in the vicinity of the substrate seems to be stronger than polymer-surface interaction. Even when the surface coverage with fibers formed by PBLG-300K is ∼70% (as shown in Figure 1a), PBLG20K molecules prefer to bind to the surface-attached fibers instead of filling the voids in a fiberlike layer. A reduction in the amount of PBLG-20K in the solution to the mass ratio shorter-to-longer polymer 1:17.5 (1:1 molar ratio) results in the formation of large fibers shown in Figure 3a. The surface density of PBLG-300K reduces dramatically, although its concentration in the solution (cPBLG-300K ) 5 × 10-5 g/mL) is sufficient to provide a significant surface coverage (similar to that shown in Figure 1a when the individual polymer was adsorbed from the solution). Fibers lose their well-defined shape, and their thickness increases from 140 ( 10 to ∼350 ( 40 nm. The softness of the surface assemblies suggests that these (11) Gupta, A. K. Biopolymers 1976, 15, 1543. (12) Variation in the length of the side chain can strongly influence intermolecular interaction between the surface-attached molecules and molecules next to the surface. In experiments on self-assembly of poly(alkyl glutamate)s with the varying length of the side chain, a change in the structure of the second layer was observed when the length of the side chain was sufficiently long. To be published.
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Langmuir, Vol. 14, No. 19, 1998 5571 Table 1. Coefficients of Diffusion and Estimated Aggregation Numbers of PBLG Molecules in 1,4-Dioxane Solution
Figure 3. Structure of PBLG layers obtained by self-assembly from dioxane solutions of PBLG-300K with a reduced amount of PBLG-20K: (a) cPBLG-300K ) 9 × 10-5 g/mL, cPBLG-20K ) 5 × 10-6 g/mL; (b) cPBLG-300K ) 5 × 10-5 g/mL, cPBLG-20K ) 9 × 10-7 g/mL; (c) cPBLG-300K ) 3 × 10-5 g/mL, cPBLG-20K ) 1.5 × 10-7 g/mL.
fiberlike aggregates consist of both PBLG-300K and PBLG-20K. The increase in fiber thickness is presumably due to the incorporation of shorter molecules in fibers by both side-to-side and end-to-head interaction, which leads to poor control of the directionality of fiber growth. In addition, an interpetation of fiber formation as a transition from individual globular molecules collapsed on the surface into clusters of unfolded rigid molecules1 requires a complementarity between associating molecules. Recent
polymer solution
polym concn (g/mL)
1012Dtr (m2/s)
Θrot (s-1)
Naggr
PBLG-300K PBLG-300K and PBLG-20K
1.1 × 10-3 1.1 × 10-3 5.9 × 10-5
6.5 ( 0.2 6.5 ( 0.2
1600 ( 150 1600 ( 150
2-3 2-3
discussion of the folding/unfolding process in proteins by analogy with polymer crystallization from solutions13 supports this argument. To study whether the association of shorter and longer molecules is a surface-driven effect or whether it occurs in the bulk solution, dynamic light scattering experiments were performed for the solution of the individual PBLG300K and the solution of the mixture PBLG-300K-PBLG20K at the weight ratio short-to-long polymer 1:18.4. To exceed the lower limit of the sensitivity of the DLS method, the polymer concentration in the solution was ∼10 times higher than that used AFM experiments. Table 1 presents the diffusion coefficients and estimated aggregation number of PBLG-300 K in the solution of the individual polymer and in the solution with the added PBLG-20K. From the values of the translational and rotational diffusion coefficients incorporated in Broersma relations,1,14 it can be suggested that some side-to-side association of macromolecules (2-3 molecules) occurs at cPBLG-300K ) 1.1 × 10-3 g/mL. Also some flexibility of PBLG-300K rods in dioxane solution cannot be ruled out.15 The scattering pattern of the solution containing PBLG300K and PBLG-20K at an equimolar ratio shows no difference compared to the individual solution of PBLG300K. The values of the translational and rotational diffusion coefficients of long-chain molecules are very close, as shown in Table 1. Since these results were obtained at a higher total polymer concentration in the solution than that used in studies of PBLG self-assembly, we can conclude that no appreciable association of the long and short molecules occurs in the bulk, and that their interaction takes place in the layer adjacent to the surface. Further reduction of the mass ratio long-to-short polymer to be as small as ca. 1:50 or even 1:200 (molar ratio 1:3.3 and 1:13.4, respectively) significantly enhances the association of the surface-attached filaments of PBLG300K. Figure 3 exhibits bundles consisting of 3-6 individual fibers obtained at the concentration of PBLG20K in the solution 9 × 10-7 g/mL and 1.5 × 10-7 g/mL, respectively. The structure shown in Figure 3 contrasts with that formed by the individual PBLG-300 K (Figure 1a), although the concentration of the long-chain macromolecules is almost the same, i.e., 3 × 10-5 g/mL. Enhanced fiber association can be attributed to the “bridging” effect of the PBLG-20K chains bound end-toend with the molecules of PBLG-300K aggregated in clusters. Association induced by shorter macromolecules does not influence surface coverage on mica, presumably because of the incorporation of all the shorter molecules associated in PBLG-300K fibers. In conclusion, we have shown that the presence of a small amount of lower molecular weight polypeptides in a solution can significantly influence the fibrogenesis of longer molecular weight chains. Although PBLG-20K is a fiber-forming polymer, in relatively small amounts it distorts the fibrogenesis of longer macrochains. Fibers (13) Subarina, J. A. Trends Polym. Sci. 1997, 5, 321. (14) (a) Jakesˇ, J.; Czech, J. J. Phys., Part B 1988, 38, 1305. (b) Schille´n, K.; Brown, W.; Johnsen, R. M. Macromolecules 1994, 27, 4825. (15) Kubota, K.; Chu, B. Biopolymers 1983, 22, 1461.
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consisting of a mixture of long- and short-chain polymers have dimensions significantly larger than those of fibers formed by individual polymers. At a concentration in the solution about 2 orders of magnitude lower than that of PBLG-300K, the lower-molecular weight PBLG-20K induces lateral association of surface-attached fibers formed predominantly from a longer polymer. This effect can have great importance for fiber formation, particularly
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for fibrogenesis of proteins in the presence of shorter polypeptide sequences. Acknowledgment. The authors gratefully acknowledge financial support from NSERC Canada through Grant 72009897. LA980455Q
