Research ArticleMATERIALS SCIENCE

Exploiting mammalian low-complexity domains for liquid-liquid phase separation–driven underwater adhesive coatings

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Science Advances  23 Aug 2019:
Vol. 5, no. 8, eaax3155
DOI: 10.1126/sciadv.aax3155
  • Fig. 1 Engineering mammalian LC domains for protein-based strong underwater adhesives through LLPS, followed by maturation into nanofibers.

    (A) Schematic of TDP43, a human DNA binding protein, which, along with specific RNAs, forms TDP43 ribonucleoprotein granules that mediate cellular processes in neuron cells. TDP43 contains both RNA recognition motifs (RRMs) and a LC domain, which is an intrinsically disordered domain enriched in glycine and uncharged polar amino acid residues. The LC domain contributes to the self-assembly of ribonucleoprotein granules, which is driven by a process termed LLPS. The featured α helix domain and aromatic residues in TDP43 LC sequence are highlighted below. (B) Schematic of the adhesive protein Mfp5 that functions in the adhesion plaques of marine mussels. Mfp5, a mussel adhesion foot protein rich in lysine and DOPA residues, displays a random coil structure in solution and is essential for the interfacial underwater adhesion of mussels. (C) The modular genetic design of adhesive proteins is enabled by rationally fusing sequences encoding the protein domains shown in (A) and (B) to form a His-tagged TLC-M fusion protein. (D) TLC-M fusion protein monomers in solution can assemble into condensed liquid-like droplets driven by LLPS (left). The liquid-like droplets tend to spread over and adsorb on surfaces, facilitating a priming process for the TLC-M coating (middle left). In a follow-up maturation process, the liquid-like droplets are locally enriched near the substrate surface. These droplets have high concentrations of protein monomers, thus promoting the formation of protofibrils on the substrate (middle right). Additional monomers can then aggregate on these surface-localized protofibrils, eventually forming amyloid nanofiber coatings on the surface. The TLC-M nanofiber coatings exhibit strong underwater adhesion owing to their large surface area and the adhesive properties of the Mfp5 domains, which are exposed at the surface of the nanofibers, external to the amyloid core.

  • Fig. 2 Morphological and structural characterization of LLPS-induced TLC-M adhesive coatings.

    (A) Photographs of adhesive coatings (top left) produced by incubating TLC-M monomer solutions at 4 and 25°C and staining of the corresponding samples with CR (lower left). X-ray fiber diffraction pattern (right) of TLC-M nanofibers of two samples showed a typical diffraction pattern of cross-β spine of amyloid nanofibers in which the meridional reflection is ~4.67 Å (corresponding to the spacing between β strands within each layer of β sheets in the fibril) and the equatorial reflection is ~9.27 Å (corresponding to the intersheet packing distances between each layer of β sheets in the fibril). (B) Morphological characterization of the hierarchical structure of TLC-M coatings formed at 4°C. TLC-M coatings were dense nanofiber meshes [transmission electron microscopy (TEM) image; left] formed from nanofiber bundles that assembled via the lateral stacking of amyloid nanofibers (TEM image; middle). These nanofibers are about 3 nm in diameter [atomic force microscopy (AFM) image; right]. (C) Thioflavin T (ThT) fluorescence curve of TLC-M in solutions (2 mg/ml) incubated at 4 and 25°C. A ThT fluorescence assay was applied to monitor and quantify the kinetics of amyloid nanofiber formation. (D) Turbidity-time curve of TLC-M in solution (1 mg/ml) incubated at 4 and 25°C, measured at optical density at 600 nm (OD600). (E) Photographs of TLC-M solutions (1 mg/ml) incubated at 4 and 25°C taken at different time intervals. (F) Differential interference contrast (DIC) microscopy images of phase separation–induced assembly of TLC-M protein monomers adsorbed on a glass surface at 4 and 25°C assessed after 1, 3, and 12 hours of incubation. LLPS-driven formation of liquid-like droplets happens during the first 6 hours, followed by the appearance of protofibrils in the vicinity of the liquid-like droplets. TLC-M monomers then aggregate on the protofibrils, eventually producing dense TLC-M nanofiber coatings. (G) Frequency change comparison in quartz crystal microbalance with dissipation (QCM-D) experiments showing the different adsorption capacities of TLC-M solutions (1 mg/ml) to a gold surface at 4 and 25°C (top). Plots of ΔD versus ΔF (bottom) corresponding to the curve shown at the top. The ΔDF value indicates the stiffness of the coatings: Higher ΔDF values suggest softer materials. ThT and turbidity data show means ± standard error of the mean (s.e.m.) of three replicate samples. ppm, parts per million.

  • Fig. 3 Underwater adhesion performance of adhesive coatings made of mammalian LC domain proteins.

    (A) Schematic of a colloidal AFM probe used to measure the asymmetric adhesion of adhesive coatings on smooth mica surfaces. (B) Comparison of adhesion forces [normalized force (F/R) and adhesion energies (Ead = F/3πR)] for TLC-M coatings produced at 4 and 25°C (measured with a gold probe tip). Representative adhesion force-distance curves in the right panel were collected on one spot of the coated mica surface using the single force mode (gold probe tip). (C) Comparison of adhesion forces and adhesion energies for the TDP43 LC domain (control), unmodified TLC-M, and DOPA-modified TLC-M coatings produced at 4°C (gold probe tip) (left) and representative adhesion force-distance curves of the control TDP43 LC, unmodified TLC-M, and DOPA-modified TLC-M coatings (right); the curves correspond to three random spots of the coated mica surface (single force mode; gold probe tip). (D) Frequency change comparison in QCM-D experiments showing the different adsorption capacities of unmodified TLC-M and DOPA-modified TLC-M coatings for a gold surface at 4°C. Inset: Plots of ΔD versus ΔF corresponding to the frequency change curve. (E) CR staining of DOPA-modified TLC-M coatings (produced in solution pH 5, 0.05 M NaCl) after a continuous 7-day incubation under harsh conditions (pH 3 and 11 buffers, 1.0 M high NaCl concentration buffer). (F) Adhesion forces and adhesion energies for DOPA-modified TLC-M coatings produced at a range of pH values (3 to 11) at 4°C measured in pH 5 buffer with a gold probe tip. (G) Adhesion forces and adhesion energies for DOPA-modified TLC-M coatings produced at a range of NaCl concentrations (50 to 1000 mM) at 4°C measured in pH 5 buffer with a gold probe tip. **P < 0.01, Student’s t test. Error bars indicate the SD. For each comparison in (B), (C), (E), and (F), n = 25 (five spots per mica plate, with each spot sampled five times using single force mode). In (B) and (D), the adhesion force curves are plotted as force-displacement curves: The x axis labeled as Z snsr (Z sensor) represents the displacement between the sample surface and the resting position of the cantilever (rather than the actual distance between the sample surface and the AFM tip). Note that all the underwater adhesion measurements were performed at 25°C aqueous temperature.

  • Fig. 4 Applications of LLPS-induced TLC-M adhesive coatings.

    (A) Digital camera images were taken under 250-nm UV light for uncoated (left) and TLC-M–coated Teflon substrates (right). The inset was water contact angle analysis, and the bottom was red quantum dot (QD) adsorption on corresponding substrates. (B) SEM sectional image of a continuous layer of LLPS-induced TLC-M adhesive coating formed on Teflon (top) and corresponding x-ray photoelectron spectroscopy (XPS) showing the distinctive element signals representative of N, O, and C (from protein structures) in the coated Teflon substrate, in contrast with the dominant F and C signals shown in the bare Teflon. A.U., arbitrary unit. (C) UV-illuminated photograph (top) and three-dimensional (3D) confocal micrograph (bottom) of a Teflon pipe showing red QDs adsorbed on the interior walls of the TLC-M–coated pipe. (D) Schematic (top), UV-illuminated photograph (middle), and 3D confocal micrograph (bottom) of a microfluidic device with a TLC-M–coated channel that was posttreated with red QDs. (E) Schematic illustration showing the repair of damage (scratch) on a Teflon substrate. We coinjected purified TLC-M monomers in solution with nonsticky fluorescent spherical PS microspheres around the damage site. Upon incubation at 4°C for 12 hours, an LLPS-induced TLC-M coating formed on the substrate and on the surface of the PS microspheres, acting as a glue that aggregated the microspheres to each other and with the substrate surface, thereby retaining them in place and filling the damage site (right). (F) Photograph (top) and SEM (bottom) images showing the damaged Teflon substrates before and after repair made using LLPS-induced coatings. Photograph of the repaired sample is taken under UV light (254 nm). A zoomed-in SEM image shows that the microspheres were piled up and glued together in the damage site.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaax3155/DC1

    Fig. S1. CR and NBT staining of self-assembled amyloid nanofibers.

    Fig. S2. Morphology of self-assembled amyloid nanofibers and phase separated liquid-like droplets of TLC-M.

    Fig. S3. Protein concentration effect on kinetics of liquid-like droplet settling and amyloid assembly.

    Fig. S4. X-ray fiber diffraction, ThT fluorescence curves, and turbidity-time curves of TDP43 LC.

    Fig. S5. 3D histogram showing specific parameters that influence the formation of liquid-like droplets.

    Fig. S6. Frequency change comparison and ΔFD curves for different protein samples tested in QCM-D experiments.

    Fig. S7. Force-distance curve tested by colloidal probe AFM.

    Fig. S8. LLPS-driven DOPA-modified TLC-M coatings on diverse of substrates.

    Table S1. Gravity effect on coacervate adsorption toward a given surface.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. CR and NBT staining of self-assembled amyloid nanofibers.
    • Fig. S2. Morphology of self-assembled amyloid nanofibers and phase separated liquid-like droplets of TLC-M.
    • Fig. S3. Protein concentration effect on kinetics of liquid-like droplet settling and amyloid assembly.
    • Fig. S4. X-ray fiber diffraction, ThT fluorescence curves, and turbidity-time curves of TDP43 LC.
    • Fig. S5. 3D histogram showing specific parameters that influence the formation of liquid-like droplets.
    • Fig. S6. Frequency change comparison and ΔFD curves for different protein samples tested in QCM-D experiments.
    • Fig. S7. Force-distance curve tested by colloidal probe AFM.
    • Fig. S8. LLPS-driven DOPA-modified TLC-M coatings on diverse of substrates.
    • Table S1. Gravity effect on coacervate adsorption toward a given surface.

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