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Flat Drops, Elastic Sheets and Microcapsules by Interfacial Assembly of a Bacterial Biofilm Protein, BslA Gilad Kaufman, Wei Liu, Danielle M. Williams, Youngwoo Choo, Manesh Gopinadhan, Niveditha Samudrala, Raphael Sarfati, Elsa C. Y. Yan, Lynne Regan, and Chinedum O. Osuji Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03226 • Publication Date (Web): 02 Nov 2017 Downloaded from http://pubs.acs.org on November 3, 2017
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Flat Drops, Elastic Sheets and Microcapsules by Interfacial Assembly of a Bacterial Biofilm Protein, BslA Gilad Kaufman†, Wei Liu‡, Danielle M. Williams¶, Youngwoo Choo†, Manesh Gopinadhan†, Niveditha Samudrala†, Raphael Sarfati§, Elsa C. Y. Yan, Lynne Regan¶, and Chinedum O. Osuji†* † Department of Chemical and Environmental Engineering, Yale University, New Haven CT 06511 ‡ Department of Chemistry, Yale University, New Haven CT 06511 ¶ Department of Molecular Biophysics and Biochemistry, Yale University, New Haven CT 06511 § Department of Physics, Yale University, New Haven CT 06511 KEYWORDS: microcapsules; protein surfactant; BslA; microfluidics
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ABSTRACT
Protein adsorption and assembly at interfaces provides a potentially versatile route to create useful constructs for fluid compartmentalization. In this context, we consider the interfacial assembly of a bacterial biofilm protein, BslA, at air-water and oil-water interfaces. Densely packed, high modulus monolayers form at air-water interfaces leading to the formation of flattened sessile water drops. BslA forms elastic sheets at oil-water interfaces leading to the production of stable monodisperse oil-in-water microcapsules. By contrast, water-in-oil microcapsules are unstable but display arrested rather than full coalescence on contact. The disparity in stability likely originates in a low areal density of BslA hydrophobic caps on the exterior surface of water-in-oil microcapsules, relative to the inverse case. In direct analogy with small molecule surfactants, the lack of stability of individual water-in-oil microcapsules is consistent with the large value of the hydrophilic-lipophilic balance (HLB number) calculated based on the BslA crystal structure. The occurrence of arrested coalescence indicates that the surface activity of BslA is similar to that of colloidal particles that produce Pickering emulsions, with the stability of partially coalesced structures ensured by interfacial jamming. Micropipette aspiration and flow in tapered capillaries reveal intriguing reversible and non-reversible modes of mechanical deformation, respectively. The mechanical robustness of the microcapsules, the ability to engineer their shape, and to design highly specific binding responses through protein engineering suggest that these microcapsules may be useful for biomedical applications.
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INTRODUCTION
Proteins perform myriad functions in biological systems and thereby inspire applications as technologically useful materials. Protein adsorption and assembly at interfaces are of particular interest for mediating interactions between inorganic and biological materials,1, 2 for catalysis, 3, 4 and for sensing.5 Properly selected proteins can also provide structural integrity for fluid compartmentalization to fabricate proteinosomes, or microcapsules, with the possibility to tailor the chemical affinity and release characteristics of the resulting constructs. These objects are deemed useful in contexts ranging from biotechnology to cosmetics and food science. Important progress has been made in recent years. Proteinosomes based on protein-polymer conjugates of bovine serum albumin and poly(N-isopropylacrylamide) have been demonstrated by Mann et al.6, 7 and stabilization of water-in-water emulsions by amyloid fibrils by Shum et al.8 Work by Lee, Hammer et al. produced stable microbubbles as biocompatible acoustic contract agents using a recombinant oleosin mutant that was engineered to improve the poor water solubility of the native form.9 Böker et al. used fungal hydrophobins to emulsify oil droplets and thereafter template the mineralization of hydroxyapatite on the droplet exterior, resulting in the formation of novel protein-mineral hybrid microcapsules.10 Other protein-stabilized emulsions mimicking Pickering behavior have also been reported.11-13 All told however, there are remarkably few examples of successful protein-based fluid compartmentalization. The poor water solubility of many surface active proteins is a strong contributing factor to the dearth of such examples.
Biofilm surface layer protein A (BslA, protein ID 4BHU) is a low molecular weight (19.1 kDa) amphipathic protein with a hydrophilic Ig domain and a hydrophobic cap.14 It has an
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overall prolate shape, as shown schematically in Figure 1A. BslA aids the assembly, and contributes to the hydrophobicity, of the biofilm matrix in Bacillus subtilis.14 In contrast to other amphiphatic proteins such as fungal hydrophobins and wild-type oleosin, the hydrophobic cap of BslA is dynamic – the hydrophobic side chains are buried in a random coil conformation in aqueous media, but form β-sheets at appropriate amphiphilic interfaces, leading to the formation of highly ordered and tightly packed monolayers.15 This structural metamorphism has an important implication as it provides stability against aggregation in aqueous suspension, yet permits assembly at interfaces. BslA forms elastic films due to monolayer formation at air-water interfaces
15-17
and emulsifies oil droplets in water.18 Genetically engineered BslA incorporating
SpyTag retains its monolayer formation properties while permitting highly specific attachment of species bearing the conjugate SpyCatcher moiety.19
Here we study BslA assembly at various interfaces and specifically exploit its interfacial assembly to
fabricate
mechanically robust
microcapsules
in
a
single
step
using
polydimethylsiloxane (PDMS) microfluidic devices. This process mirrors single-step microcapsule fabrication by interfacial complexation as recently advanced20-22 but has the added benefit of involving only a single-component, rather than requiring conjugate species. Stable, monodisperse oil-in-water BslA microcapsules could be readily produced, whereas water-in-oil systems displayed arrested coalescence. The disparity in microcapsule stability is attributed to the shape asymmetry of BslA. This shape asymmetry is reflected by the large ratio of hydrophilic to hydrophobic components of the protein which predisposes the stabilization of oil-in-water, rather than water-in-oil structures, in direct analogy to small molecule surfactants. Oil-in-water microcapsules displayed irreversible deformation due to interfacial jamming of BslA, analogous
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to that seen in Pickering emulsions. These characteristics point to a particle-like behavior of BslA in stabilizing oil-water interfaces. BslA monolayer formation at air-water interfaces leads to flattening of sessile water drops, due to the cohesive energy of the BslA film which manifests as a large bending stiffness relative to the water’s surface tension. Electron microscopy and Xray measurements show that the monolayer consists of a densely-packed pseudo-crystalline arrangement of BslA. Overall, this work highlights the assembly of BslA across multiple interfaces, explores the structure and properties of the resulting interfacial structures, and documents the mechanical properties and stability of constructs, bubbles and microcapsules, formed by BslA interfacial assembly.
EXPERIMENTAL SECTION
Expression and purification of BslA(29-176)
DNA encoding BslA29-176 was synthesized by GenScript and cloned into the pGEX-6P-1 vector using BamHI and XhoI restriction sites. The resulting plasmid was then transformed into E.coli strain BL21(DE3). The cells were cultured at 37 °C in 500 mL of Lysogeny broth (LB) containing 100 µg/ml of ampicillin, and induced with 250 µM of isopropyl β-thiogalactoside (IPTG) at an optical density (600 nm) OD600 of 0.6~0.8. The cells were then grown at 25 °C for overnight expression, spun down and the pellets were stored at -80 °C. For purification, the cells were resuspended in the lysis buffer (25 mM Tris-HCl, pH 7.4 and 250 mM NaCl) in the presence of EDTA-free Complete Protease Inhibitor (Roche). The cells were lysed by sonication and the cell debris were removed by centrifugation at 13,000 rpm at 4 °C. 1 ml of Glutathione
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Sepharose 4B beads (GE Healthcare) were added to the clear lysate and the sample was nutated for 2-3 hrs at 4 °C to allow for sufficient binding. The protein-bound beads were then collected by centrifugation, followed by first wash with the lysis buffer and second wash with the cleavage buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol). Next, 20 µL of PreScission protease in 3 mL of cleavage buffer were added to the beads and the sample was kept on a nutator for overnight cleavage at 4 °C. The solution was collected and the remaining impurities were removed by gel filtration using Superdex 75. The purity of the protein was confirmed by SDS-PAGE. For all other experimental characterization, purified BslA was concentrated using centricons and buffer-exchanged into storage buffer (10 mM phosphate buffer, pH 7.4 and 100 mM NaCl). The final concentration of BslA stock solution produced in this manner was 40 µM.
Surface pressure-area isotherm and compression modulus isotherm
Surface pressure-area isotherms were obtained using a KSV mini Langmuir trough (KN2002, KSV Instruments Ltd, Finland). Two symmetric Telfon barriers were controlled by KSV Nima software and a Wihelmy plate was used to record the surface pressure during compression. The surface pressure was recorded by the Wihelmy plate versus the mean molecular area, which was calculated by the software assuming all molecules added stay within the area between two barriers. All experiments were conducted in a room with at a constant temperature controlled at 20 ± 1 °C.
Microfluidic fabrication and pipette aspiration
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Microcapsules were fabricated in microfluidic devices constructed from polydimethylsiloxane (PDMS) molds bonded to glass surfaces, or using t-junction devices produced from microtubing. PDMS microfluidic devices were produced using standard soft lithography fabrication, with fluid flow controlled by a syring pump (KD Scientific KDS200). Micropipette aspiration experiments were conducted by application of suction pressure using a custom apparatus mounted on an optical microscope, as previously described.23
RESULTS AND DISCUSSION
Film Formation at Air-Water Interfaces
We examined BslA assembly at an air-water interface by visually monitoring the behavior of a sessile water droplet containing 0.06 mg/ml of the protein. The droplet was placed on a hydrophobic surface, silicon substrate treated with trichloro(1H,1H,2H,2H-perfluorooctyl)silane, that suppressed fluid spreading. Initially the drop had a uniformly curved surface, Figure 1B, as expected for a fluid interface with a finite surface tension. After about 5 minutes however the top surface of the drop showed a pronounced flattening, Figure 1C. This flattening indicates that the BslA monolayer that develops at the air-water interface has solid-like behavior and resists bending to a large enough extent to overcome the area minimizing influence of surface tension. Wrinkles are easily seen in bright field optical micrographs of partially dried droplets (Supporting Information, Figure S1) providing additional evidence that a solid-like membrane forms at the air-water interface. The modification of interfacial curvature associated with flat
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droplets has been observed for hydrophobin HFBI24-26, synthetic peptides27, 28, and, recently, for polydopamine stabilized droplets.29 Our observation marks the first demonstration of this phenomenon in BslA and suggests that BslA monolayers at air-water interfaces have considerable stiffness, or bending rigidity. We did not observe flattening of the sides of the droplets. The reasons for preferential flattening at the droplet top versus droplet sides are unclear. One possibility is that buoyancy plays a role in BslA accumulation and film formation, as reported by Yamasaki and Haruyama in the case of HFBI hydrophobins.26 Another possibility is that spatial differences in the droplet curvature promotes the initiation of flat film formation at the droplet apogee which has a smaller curvature compared to the droplet side, for contact angles larger than 90 º. This resonates with the observations of Jang, Lee et al. that the kinetics of faceting in droplets due to peptide assembly varies inversely with droplet curvature.28
Figure 1. (A) Schematic representations of BslA structure in ribbon and cartoon form. (B-D) Photographs and schematics (inset) showing time evolution of a droplet of an aqueous solution of BslA (0.06 mg/ml) deposited onto a hydrophobized silicon wafer. (B) Immediately after placing the drop on the silicon surface the drop has a contact angle of roughly 115 °, t=0 min. ; (C) t=5 min. The contact angle decreased slightly, likely due to protein adsorption onto the hydrophobic substrate, and stabilized around roughly 90 °.; (D) t=10 min. The inset schematics show BslA assembly into a flat film at the top of the droplet as this is where flattening was observed. This does not imply however that the sides of the droplets are not coated in BslA, but simply that the droplet sides do not exhibit flattening. Scale bar is 0.5 mm.
Surface pressure measurements were conducted using a Langmuir-Blodgett apparatus to characterize the surface activity and mechanical properties of the BslA monolayer. The surface
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pressure isotherm, Figure 2, displays a collapse pressure of ∼65 mN/m and a limiting mean molecular area ~ 700 Å2 per molecule. The collapse pressure is larger than that for typical macroscopic protein films30 (~25-35 mN/m) but similar to that of common phospholipids31, 32 as well as recently explored polystyrene and Janus-particle monolayers.33,
34
The modest ratio
between the limiting area and the estimated molecular cross-sectional area (500 Å2), and the large collapse pressure both point to the formation of highly ordered and mechanically robust membrane monolayers. This is corroborated by the area compression modulus of the monolayer, ܥ௦ , (inset, Figure 2), which is extracted from the surface pressure isotherm as a function of surface pressure, ߨ, as
ܥ௦ = − ܣ൬
݀ߨ ൰ ݀ܣ
where ܣis the mean molecular area at the given surface pressure. ܥ௦ is numerically equivalent to the membrane area expansion modulus, ܭ . The compression modulus is in the range of 100470 mN/m for physically relevant surface tensions and is similar in scale to the compression modulus (
