The Drosophila Transcription Factor Ultrabithorax Self-Assembles into

Biomacromolecules 2009, 10, 829–837

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The Drosophila Transcription Factor Ultrabithorax Self-Assembles into Protein-Based Biomaterials with Multiple Morphologies Alexandra M. Greer,†,⊥ Zhao Huang,†,⊥ Ashley Oriakhi,† Yang Lu,‡ Jun Lou,‡ Kathleen S. Matthews,† and Sarah E. Bondos*,†,§ Departments of Biochemistry and Cell Biology and Mechanical Engineering and Materials Science, Rice University, 6100 South Main Street, Houston, Texas 77005, and Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, 440 Reynolds Medical Building, College Station, Texas 77843-1114 Received November 14, 2008; Revised Manuscript Received February 11, 2009

The use of proteins as monomers for materials assembly enables customization of chemical, physical, and functional properties. However, natural materials-forming proteins are difficult to produce as recombinant protein monomers and require harsh conditions to initiate assembly. We have generated materials using the recombinant transcription factor Ultrabithorax, a Drosophila melanogaster protein not known or anticipated to form extended oligomers in ViVo. Ultrabithorax self-assembles at the air-water interface into nanoscale fibers, which further associate to form macroscale films, sheets, ropes, and tethered encapsulates. These materials self-adhere, allowing construction of more complex architectures. The Ultrabithorax sequence contains two regions capable of generating materials, only one of which contains motifs found in elastomeric proteins. However, both minimal regions must be included to produce robust materials. Relative to other protein-based materials, Ultrabithorax assembles at significantly reduced concentrations, on faster timescales, and under gentler conditions, properties that facilitate future materials engineering and functionalization.

Introduction Protein-based materials have the potential to be customized for a variety of applications, including drug delivery, tissue engineering, surgical sealants, medical imaging, biosensors, bionanofabrication, and biomineralization.1,2 However, realization of this promise requires development of a variety of materials with different chemical, mechanical, and functional properties.3 For instance, macroscale materials in medical applications must be biodegradable,4-6 biocompatible,7,8 and have mechanical properties matching the tissues of interest,9,10 whereas materials destined for bionanofabrication must form rigid nanoscale three-dimensional structures.11,12 Consequently, the development of a variety of protein-based materials is required to meet these diverse needs. Effective production of engineered materials poses a second challenge. Sequence engineering and functionalization is limited by length for in Vitro synthesized peptides and is prohibitively difficult for materials derived ex ViVo. For medical applications, natural materials extracted ex ViVo could potentially transfer a disease from the organism to a patient.13 These problems could be solved by assembling materials from recombinant monomers produced in Escherichia coli; however, recombinant monomers often require high temperatures, exposure to organic chemicals, or extreme pH to stimulate protein assembly14-18 (Supporting Information Table 1). Such harsh processing would likely * Corresponding author. E-mail: [email protected]; phone: 979-845-5399; fax: 979-847-9481. † Department of Biochemistry and Cell Biology, Rice University. ⊥ These authors contributed equally to this work. ‡ Department of Mechanical Engineering and Materials Science, Rice University. § Texas A&M Health Science Center.

preclude incorporation of many active heterologous proteins and thus limit functionalization of the material by genetic methods. By contrast, we have devised a method to produce nanoscale and macroscale materials - without treatment with high temperature, acids, or organic solvents - from recombinant protein produced in E. coli. These materials are composed of Ultrabithorax (Ubx), a Drosophila melanogaster Hox transcription factor. During animal development, Hox proteins instigate developmental programs to differentiate serially repeated regions into distinct body structures.19,20 As transcription factors, Hox proteins bind DNA via their homeodomain and subsequently activate or repress transcription through heteromeric protein interactions.21-27 We have generated ordered Ubx materials in a variety of complex morphologies, several of which resemble materials formed by elastomeric proteins in Vitro or in ViVo. Under gentle conditions, Ubx self-assembles into fibers within 2 h at protein concentrations 2 orders of magnitude less than required for assembly of elastomeric protein materials. Surprisingly, examination of Ubx truncation mutants revealed two minimal materials-forming regions within Ubx, one of which lacks sequence features found in either amyloidogenic peptides or elastomeric proteins. Since both minimal regions are required to generate robust materials, the molecular interactions underlying materials assembly may be unique. These materials thus provide a novel building block for facile engineering and functionalization of protein-based materials.

Experimental Section Plasmid Construction of Ubx Deletion Mutants. Ubx splicing isoform Ia (herein termed “Ubx”) and Ubx variants were cloned between the NdeI and BamHI sites in the pET19b vector (Novagen),

10.1021/bm801315v CCC: $40.75  2009 American Chemical Society Published on Web 03/18/2009

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Table 1. Dependence of Rope Length and Formation on Ubx Concentration, Measured by Drawing Five Ropes at Each Concentration of Ubx after Incubation at Room Temperature for 2 h to Generate the Average and Standard Deviation Values for Rope Length Ubx concentration (µg/mL)

rope length (mm)

500 300 150 75 50 25

15 ( 7 10 ( 4 7(3 3(2 1(1 0

which appends a His-tag to the N-terminus of Ubx. The constructs expressing Ubx1a or N-terminal truncations were the gift of Ying Liu (Rice University).21 To generate C-terminal truncations, two consecutive stop codons were inserted into the Ubx coding region using the QuikChange site-directed mutagenesis kit (Stratagene). Protein Expression and Purification. Plasmid constructs were transformed into BL21 (DE3) pLysS E. coli cells. E. coli cultures were cultivated in Luria broth plus 50 µg/mL carbenicillin and 30 µg/mL chloramphenicol (LB) at 37 °C unless otherwise stated. For expression, 10 mL of an overnight culture, inoculated from a single colony, was used to inoculate a 1 L LB culture, which was grown to an optical density at 600 nm of 0.6-0.8. Cell cultures were then cooled to 30 °C, and Ubx expression was induced with 1 mM IPTG prior to a further 105 min of fermentation. Cells were harvested by centrifugation at 7000g for 15 min and stored at -20 °C in aliquots corresponding to 1 L of culture. Each aliquot was thawed at room temperature and lysed in 20 mL of lysis buffer (50 mM sodium phosphate buffer, pH 8.0, 5% glucose w/v, 500 mM NaCl, 1 protease inhibitor tablet (Roche), 0.8 mg/L DNase I). Cell lysates were centrifuged at 18000g for 20 min. The supernatant was loaded on a nickel-nitrilotriacetic acid (NiNTA) agarose resin column (Qiagen), which was pre-equilibrated with 20 mL of equilibration buffer (5% glucose w/v, 500 mM NaCl, 50 mM sodium phosphate buffer, pH 8.0). The column was then washed by 10 column volumes of W1 buffer, 10 column volumes of W2 buffer, and 5 column volumes of W3 buffer (Equilibration Buffer containing 20 mM, 40 mM, and 80 mM imidazole, respectively). Protein was eluted at greater than 80% purity with 10 mL of elution buffer (200 mM imidazole dissolved in equilibration buffer) (Supporting Information Figure 1). The concentrations of the purified Ubx samples were determined using the BioRad protein assay (BioRad). Approximately 2 mg of dithiothreitol (DTT) was added to each 1 mL elution volume to maintain the protein in the reduced state. Purified Ubx was dialyzed into freezing buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 5% glucose, 1 mM DTT pH 7.5) before storage at -80 °C. Production of Ubx-Based Materials. Ubx Film. Ubx was diluted to 0.6 mg/ml, unless otherwise stated, using elution buffer. Although Ubx forms films at much lower concentrations (Table 1), several of the trunctation mutants required higher concentrations to generate materials, and thus all experiments used 0.6 mg/mL protein for consistency. Materials formed at these two concentrations exhibited no difference in appearance in electron microscopy studies. Ethylenediaminetetracetic acid (EDTA) was added to 10 mM to prevent amorphous aggregation. Ubx solution (100 µL) was placed on the surface of a siliconized coverslip at room temperature. Ubx film spontaneously forms at the air-water interface after approximately 1 h. Ubx Ropes. A needle or pipet tip was used to contact the surface of Ubx film and was withdrawn slowly to draw ropes. Ubx Sheets. Ubx protein samples were concentrated to 1 mg/mL using Vivaspin concentrators with a 10 kD cut off (Viva Science). EDTA was added to a final concentration of 10 mM. Ubx solution (100 µL) was placed at the surface of a siliconized coverslip, and covered with a 15 mL plastic tube cap to deter evaporation. Ubx sheets formed spontaneously at the air--buffer interface at room temperature overnight.

Greer et al. Ubx Lattices. The outer turn of a paper clip, in which the inside turn had been bent out of the way, was used as a support for lattice construction. Ubx ropes were wound around the parallel arms of an opened paper clip such that the ropes intersect between the supporting arms. Freshly made Ubx ropes adhere to both the metal supports and to each other, forming a lattice. Lattices maintain their form after 24 h of drying in air and subsequent removal from the supports. Ubx Bundles. A dome-shaped 20 µL drop of deionized water was placed on the surface of a siliconized coverslip. A series of Ubx ropes were placed in parallel, approximately 1 mm apart, on the paper clip support, and subsequently dipped into the drop of water. Upon slow withdrawal, interactions with the shrinking water surface pull the ropes toward the middle of the series, where the ropes adhere to each other, forming a multirope bundle. The degree to which the ropes fuse in the bundle appears to be dependent on the age of the rope. Ubx Tethered Encapsulates: Microbaskets. Ubx sheets were prepared as described. Objects with pointed ends (pipet tip, needle, etc.) were used to make contact with the surface of the sheet and slowly withdrawn to produce mini-encapsulated baskets. Ubx Tethered Encapsulates: Macrobaskets. Ubx macro-encapsulated baskets form spontaneously during extremely slow extrusion of a purified Ubx solution (g2 mg/mL) through a plastic two-way stopcock. Protein Stain. Izit Crystal Dye (Hampton Research, 1 µL) was added to 100 µL of eluted protein on a slide and left for 1-2 h for fibers to form. Ropes pulled from these drops were tinted blue, indicating the presence of protein, whereas exhausted drops (i.e., the remaining drop after rope formation) lacked blue color. Scanning Electron Microscopy (SEM) Imaging. Ubx ropes, lattices, and bundles were transferred to the surface of double-sided carbon tape attached to SEM specimen mount stubs. Ubx sheets and ropes were transferred using a needle, whereas Ubx film was carefully lifted using plastic coated wire bent into a stem-loop shape. Samples were sputter coated with gold for 1 min at 100 mA and examined either using an FEI-XL30 environmental scanning electron microscope or an FEI Quanta 400 field emission scanning electron microscope, each with a beam voltage of 200 kV and a spot size of 3. Western Blot Analysis. Ubx ropes were solubilized in 4X sample buffer (250 mM Tris, 40% glycerol, 4% SDS, 4% BME, 0.02 mg/ml bromophenol blue, pH 6.8) by mechanical disruption with a syringe needle followed by repeated cycles of heating (90 °C for 30 min.), vortexing, and sonication (1 min intervals), with the entire process requiring approximately 2 h. Ubx monomeric protein was diluted with 4X sample buffer to generate a positive control. Samples were separated using a 12% 29:1 polyacrylamide gel prior to transfer at 150 V for 20 min to a nitrocellulose membrane (Schleicher & Schuell). Ubx was detected using FP3.38 as the primary antibody28 at a 1:200 dilution in phosphate-buffered saline. Horseradish peroxidase-conjugated goat antimouse antibody (Calbiochem, 1:2000 dilution) was used as the secondary antibody. Mechanical Testing. The mechanical characterization of Ubx fibers was carried out using a Gatan Microtest tensile tester. The force sensitivity of the load cell is 0.0001N with maximum 2N load capability. The sensitivity of the extensometer is 0.001 mm. A loading speed of 0.1 mm/min and a data sampling rate of 500 ms were used. Samples were attached to clamps via Loctite 495 adhesive. Sample diameter was measured in five locations for each rope by SEM, and the average was used to calculate stress.

Results and Discussion Rapid Self-Assembly of Ubx Fibers, Films, and Sheets. Ubx is extremely prone to aggregation into amorphous flocculates.29 During the process of identifying conditions that inhibit amorphous aggregation, we fortuitously discovered Ubx spontaneously assembles into ordered aggregates in aqueous buffer. The surface of Ubx-containing buffer, incubated at room temperature for 1.5-2 h, acquires a “matte” or opaque appear-

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Figure 1. Basic materials formed in a hierarchical manner by Ubx. (A) A Ubx fiber, 50 nm in diameter (arrow). (B) Ubx film, formed from self-associating fibers. (C) Ubx sheet, generated by continuing to allow assembly after Ubx film has been formed. (D) A Ubx rope being pulled from a Ubx film. (E,F) Ubx rope at low and high magnification, respectively. (G) Ubx rope, fractured by the SEM beam, splinters into 50 nm fibers aligned along the main axis (arrow). (H) The free end of a severed rope shows a solid core.

ance beginning after ∼1 h (Supporting Information Figure 2). This altered appearance is due to spontaneous Ubx aggregation into water-insoluble fibers, approximately 50 nm in diameter, at the air-water interface (Figure 1A). These fibers selfassemble into a film that coats the liquid surface and causes the matte appearance (Figure 1B). Because the fibers interact strongly with each other, only portions of individual fibers are visible at the edges of fractured films. Consequently, both ends of a single fiber have never been observed, preventing measurement of fiber length. Ubx fibers and films have never been observed (i) in the bulk solution, (ii) in full tubes lacking air, or (iii) during overexpression of Ubx in Drosophila or E. coli. Consequently, Ubx assembly into fibers and films appears to require an air-liquid interface. Similarly, self-assembly of miniature silk proteins has also been observed at the air-water interface.30 Fiber assembly and subsequent film formation are required to generate the remaining material architectures. Continued polymerization of film generates Ubx sheets (Figure 1C), a far more robust material that can be easily lifted from the surface of the liquid without tearing. Since Ubx sheets also form at the air-water interface, their morphology is dependent on the size and shape of the sessile drop. Freshly generated Ubx sheets are extremely self-adhesive. Generation of Ubx Ropes. Ubx films can be drawn into ropes (Figure 1D). The diameter of an individual rope is uniform, provided that (i) the film has not begun to form a sheet, (ii) the protein concentration is between 0.075 mg/mL and 0.6 mg/mL (Table 1, Supporting Information Table 1), and (iii) the rope was drawn at an even speed (Figure 1E,F). Between ropes, diameters vary between 5 and 30 µm, apparently dependent on the degree to which the film polymerized prior to drawing the rope. These diameters are comparable to those observed for electrospun spider silk and silkworm silk.31 Ropes are composed of longitudinally aligned fibers (Figure 1F,G), and have a solid core (Figure 1H), similar to ropes assembled from a miniature version of a spider silk gene.30 The maximum possible rope length is proportional to the surface area of the film. Our longest rope thus far measured 7 cm and was formed at the surface of a 200 µL drop of Ubx. One liter of bacterial culture expressing Ubx is, therefore, sufficient to generate more than 1 m of rope. This procedure is similar to that described by Teule´ et al.,32 in which fibers were drawn from a spider silk-like recombinant protein. However, there are two notable exceptions. Ubx film adheres to the pipet tip, and thus does not have to be held by tweezers in order to be drawn into rope. More importantly,

Figure 2. Materials are composed of Ubx protein. (A) A Ubx rope is stained by Izit, a dye that selectively interacts with protein. (B) A western immunoblot identifies similar bands for purified Ubx (lane 1) and the protein contained within Ubx ropes (lane 2). The positions of molecular weight markers are indicated to the left.

uniform Ubx ropes can be produced at protein concentrations 2 orders of magnitude less than for the spider silk-like recombinant protein (Supporting Information Table 1). Indeed, Ubx generates amorphous aggregates at these high concentrations. Verifying Material Composition. The apparent ability of a Drosophila transcription factor to form materials prompted us to verify that these materials are, indeed, composed of Ubx. Materials do not form from buffer in the absence of Ubx. Alternatively, a component of the purified protein solution other than Ubx may form the materials, and Ubx is passively incorporated. To test this hypothesis, we performed a mock purification using E. coli cells lacking Ubx. The resulting solution does not form materials, simultaneously demonstrating that neither buffer components nor any E. coli proteins that may contaminate Ubx preparations at very low levels are sufficient to generate materials. Ropes stain blue in the presence of Izit Crystal Dye, demonstrating ropes do contain protein (Figure 2A). Denaturing gel electrophoresis was used to compare the protein in materials with purified Ubx. Two hours of heating, vortexing, and sonication in denaturing buffer were required to depolymerize these robust materials for gel electrophoresis. We observed that these materials are composed of a single protein, which is comparable in size to purified, monomeric Ubx. Western blots demonstrated the protein in the materials binds FP3.38, an antibody that interacts with the Ubx homeodomain28 (Figure 2B). FP3.38 is sufficiently specific to be used for immunohistochemistry experiments and is only known to cross-react with Abdominal A, another Drosophila Hox transcription factor with a nearly identical homeodomain sequence.28 Since recombinant Ubx is purified from E. coli and thus Abdominal A is absent, we conclude these materials are composed of Ubx protein. Heat Stability of Ubx Materials. The extreme measures required to depolymerize Ubx materials for gel electrophoresis

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Figure 3. Characterization of Ubx materials. (A) A Ubx rope, stained with Izit dye, and transferred to a phosphate-buffered saline solution attached to a syringe needle prior to heating. (B) The same Ubx rope after 1 h and (C) two hours of heating at 98 °C. Ubx ropes do not degrade at high temperatures, demonstrating surprising heat resistance. (D) Stress-strain curves, each measured using a different Ubx rope. (E) On average, Ubx ropes have an ∼53% breaking strain, and thus are more extensible than many protein-based materials.33-35

Figure 4. More complex architectures formed by Ubx materials: (A) a bundle composed of six partially melded ropes; (B) a bundle composed of two ropes which completely meld to one another; (C) a twist of two Ubx ropes; (D) the intersection of two ropes in a lattice, which forms a stable interaction; (E) a tethered microencapsulate, formed by drawing a Ubx sheet, contains approximately 0.5 µL of buffer; and (F) a macroencapsulate, which contains approximately 4 µL of buffer, and is formed by gradual extension of a hanging Ubx drop.

suggest that these structures are extremely resistant to heat. To test this hypothesis, a dried Ubx rope was stained blue with Coomassie dye to aid visualization and subsequently submerged in a phosphate-buffered saline bath at 98 °C. The rope withstood several hours of heat, indicating that exposure to high temperatures does not degrade or depolymerize the materials (Figure 3). Properties of Ubx Materials. Ubx ropes and sheets share many useful attributes. Freshly generated materials are well hydrated and extremely flexible (Figure 2A). Indeed, a Ubx rope can be stretched to an additional 53% ((19%) of their original length before fracturing, an extensibility exceeding that of collagen, silkworm silk, and spider dragline silk, but only onethird that of elastin33-35 (Figure 3D, E). The maximum stress (13 ( 7 MPa) and toughness (5.3 ( 4.0 MJ/m3) are comparable to other highly extensible protein materials.32-35 Ubx materials stiffen with drying (approximately 3 days). Desiccated materials are mechanically brittle, but durable, and can be stored at room temperature in air for years. Once dried, Ubx materials maintain their structure in the absence of supports or scaffolds. Dried ropes can be rehydrated without shearing or breaking. Recently generated Ubx materials adhere to glass, plastic, wood, metal, and Teflon. Ropes and sheets also adhere and meld

to themselves and each other, allowing generation of more complex architectures (see below). In contrast, silkworm silk fibers require additional proteins, termed “sericins”, to anneal fibroin fibers.36,37 Construction of Complex Ubx Materials: Bundles, Twists, Lattices, and Tethered Encapsulates. Because Ubx sheets and ropes are self-adhesive, they can be utilized as building blocks for either manual construction or self-assembly of materials with more complex architectures. Ubx bundles consist of aligned Ubx ropes that coalesce to form thick bundles (Figure 4A,B). These ropes fuse to varying degrees, depending on the age of the ropes upon creation of the bundle. Aligned, but not fused, ropes can also be wound to form twists (Figure 4C). Nonaligned Ubx ropes that intersect also fuse at their contact point, without exposure to water, to create Ubx lattices (Figure 4D). Again, the degree of fusion is dependent on rope age. Once the materials dry in air (overnight to one week, depending on size), these lattices stiffen and retain their original configuration when released from their support. Hanging drops of Ubx self-assemble into tethered encapsulates, or baskets, in which Ubx sheets, encapsulating liquid, are suspended from a Ubx rope. Microencapsulates, which only encase ∼1 µL or less, can be formed by attempting to pull rope


Figure 5. Schematic depicting the hierarchical assembly of many materials architectures from Ubx fibers. Ubx fibers, film, and sheets form spontaneously at the air water interface. Construction of all other structures requires external forces. Ubx ropes are drawn from film. More complex architectures (bottom row) are composed of sheets and/or ropes and are constructed by hand or by gravity.

from sheets instead of film or from higher protein concentrations (Figure 4E). Macrobaskets are formed from a hanging drop and can encapsulate 3-10 µL of liquid (Figure 4F). Macrobaskets are sufficiently robust that they do not rupture even when the encapsulate is rapidly swung in circles from the distal end of its suspending rope. Although the capsules partially collapse as the material dries, these structures are essentially stable for weeks when suspended in air. In both Ubx film and Ubx ropes, we were able to observe lateral interactions between Ubx fibers (Figure 1G). We hypothesize that further interactions between Ubx fibers enable rope, film, and sheet assembly and subsequent hierarchical construction of more complex structures, i.e., sheets, bundles, lattices, twists, and tethered encapsulates (Figure 5). Materials-Forming Conditions. Most recombinant proteins require harsh conditions to trigger materials assembly, including exposure to high temperatures, organic solvents, pH extremes, or metals14-17,38-42 (Supporting Information Table 1). Such conditions would be expected to denature and possibly aggregate many proteins.42,43 In contrast, Ubx oligomerizes in nondenaturing aqueous solution near neutral pH at either room temperature or 4 °C. Furthermore, Ubx self-assembles into fibers visible by SEM much more rapidly (2 h) than amyloidogeneic proteins, which typically require days to weeks (Supporting Information Table 1). Finally, Ubx readily forms materials at 0.075 mg/ mL, a concentration 2 orders of magnitude lower than that of most other protein-based materials (Table 1 and Supporting Information Table 1), thus reducing the stringent requirements on protein expression and purification for materials generation. Indeed, the conditions typically utilized to trigger amyloid or elastomeric protein assembly actually inhibit generation of Ubxbased materials. The unusually facile assembly of Ubx into materials reflects reduced thermodynamic barriers for materials formation relative to other protein systems. Comparison of the Ubx Amino Acid Sequence with That of Materials-Forming Proteins. Given that neither physical stimuli nor exogenous chemicals are required for materials formation, the Ubx amino acid sequence is sufficient to dictate this protein’s surprising ability to generate materials under mild conditions. Therefore, identification of the minimal region of Ubx required to generate these materials potentially yields insight into the molecular mechanisms underlying formation of these unique structures. An initial examination revealed similarities in the sequence of the N-terminal 216 amino acids of Ubx with other proteins that form filamentous materials (Figure 6A). A large fraction (>60%) of this region is composed of a disordered region greater than 100 amino acids in length21,44 (Figure 6A). Such regions potentially form amyloid easily, presumably because formation


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of amyloid precursors does not compete with folding to a stable native state.45,46 Furthermore, amyloid fibers may interact laterally with each other,47 as we have observed in Ubx film and ropes. Thus, one possibility is that this large disordered region drives Ubx assembly into amyloid-like structures. Ubx materials also share structure and sequence similarities with elastomeric materials, such as silk, elastin, and mussel adhesive proteins. Ubx ropes and macrobaskets resemble structures created from a recombinant spider silk-like protein,32 and Ubx bundles appear similar to elastin fibers purified ex ViVo from ligaments.48 The Ubx sequence includes three alaninerich or polyalanine regions, a motif common to araneoid and lepidopteran silks.49,50 Ubx also contains glycine-rich regions,21 a feature common in most elastomeric proteins15,17,49,51 (Figure 6A). The largest glycine-rich region, spanning amino acid 107 to 208 within the disordered domain, can be subdivided into four regions with distinct sequence features (Figure 6A,C). The first glycine-rich region (a.a. 107-123) includes a homoglycine repeat 13 amino acids long (a.a. 111-123). The second region (a.a. 124-146) is enriched in glycine and alanine, similar to many araneoid and lepidopteran silks.49 The middle region (a.a. 147-175) is weakly enriched in glycine and proline, although these residues never flank one another as found in many silks and in elastin.17,49 The final region (a.a. 176-209) includes a high content of glycine, valine, and alanine, a combination predicted to form amyloid rather than disorder or turns.52 Taken together, much of the amino acid content of the disordered region also bears some resemblance to that for natural elastomeric proteins. Because of the high glycine content, Ubx also contains multiple copies of amino acid motifs found in elastomeric proteins. GGX and GXXP are both repeated motifs in elastin, and GGX is also found in spider silk.50,52,53 The glycine-rich region in Ubx (amino acids 107 to 209) includes 3 GXXP motifs and 13 GGX motifs (Figure 6C). Although Ubx has far fewer of these motifs than a typical elastomeric protein, even a single elastomeric motif can undergo the structural transformations characteristic of this protein class.54 On the basis of these sequence similarities, we predicted that, if the molecular interactions underlying Ubx materials formation are similar to those for amyloidogenic and/or elastomeric proteins, then the region between amino acids 107 and 209 should be critical for materials formation. Experimental Identification of the Minimal Materials-Forming Domains. Because Ubx can be produced as a recombinant protein in E. coli, we were able to identify regions crucial for materials assembly by examining the ability of Ubx truncation mutants to form ropes. All N-terminal and C-terminal Ubx deletion mutants produce soluble, active proteins (Supporting Information Figure 1) and, for mutants encompassing the homeodomain, are capable of binding DNA.21,22 Each variant is named for the amino acids included; for instance, the Ubx19380 mutant includes a histidine tag, the initial methionine, and amino acids Ubx19-380. Boundaries for the truncation mutants bisect neither potential secondary structural elements nor evolutionarily conserved motifs21 (Figure 6B). Full length Ubx forms materials within 2 h, whereas the slowest truncation mutants capable of forming materials require 4 h at room temperature. Consequently, each protein was tested for film/ rope formation after 2 and 4 h of sessile drop incubation at room temperature. Because fiber formation must be painstakingly evaluated by SEM, we utilized rope formation as an assay for materials assembly. In addition, the length of rope that can be drawn

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Figure 6. Schematic of Ubx and Ubx deletion mutant show key sequence features. (A) Bar diagrams of the Ubx sequence schematic depict overlap between functional domains, regions containing intrinsic disorder, elastomeric motifs and regions enriched in a subset of amino acids. The transcription activation domain (AD),21 Extradenticle protein interaction motifs (YPWM and UbdA),55 the alternatively spliced microexons (ME),56 the homeodomain (HD), and a partial transcription repression domain (RD) are labeled.23,24 (B) Ubx sequence schematics of the truncation mutants utilized to locate minimal materials-forming regions. The average ropes after 2 and 4 h are listed to the left. (C) The sequence of the glycine-rich region, subdivided into four domains based on amino acid prevalence.

correlates well with whether the rope appears robust or brittle, allowing us to parse the truncation mutants by materials quality. In general, Ubx variants either generated robust materials comparable to full-length protein (g1 cm in length), or produced brittle and therefore short (e5 mm) ropes. Deleting first from the N-terminus, we found that Ubx19380 forms short, brittle ropes (Figure 6B). Surprisingly, this effect is ameliorated in Ubx49-380, Ubx88-380, and Ubx139380, which all form robust materials, although they require a longer incubation time to do so. Removal of amino acids 2-18 therefore exposes a region that impedes Ubx-Ubx interactions. Residues 19-48 either comprise this inhibition region or are required for its function, since removal of this segment restores the ability to form robust materials. Surprisingly, Ubx139-380 and Ubx216-380 both form ropes, even though these truncations remove either a portion or all of the sequences resembling elastomeric proteins. However, these truncations are difficult to draw, producing shorter, more brittle ropes than even Ubx49-380 and Ubx88-380. Ubx variants with further deletions from the N-terminus did not produce materials, marking amino acid 216 as the N-terminal boundary of the minimal materials-forming unit. Even though Ubx139-380 and Ubx216-380 both generate brittle ropes, Ubx174-380, the intermediate truncation, does not form materials. Thus, removal of residues 139-173 exposes a second region of Ubx that inhibits materials assembly. Removal of this inhibitory region in the Ubx216-380 variant restores

materials formation. Since much of the region between amino acids 173 and 215 is intrinsically disordered (Figure 6A), the motion of this highly flexible region may physically block Ubx-Ubx interactions. Indeed, monomeric Ubx174-380 also binds DNA with an affinity more than 20 times poorer than the full-length protein, an inhibition largely absent in Ubx216-380.21 Therefore, amino acids 173-215 debilitate multiple Ubx macromolecular interactions. The C-terminus of the minimal unit was identified by making progressive deletions from the C-terminal end of the Ubx216380 variant, the smallest N-terminal truncation mutant that still forms materials (Figure 6B). Ubx216-356 does form ropes, even though a polyalanine region, a key motif in spider silks, was removed. By contrast, Ubx216-344, which additionally removes a portion of the DNA-binding homeodomain (Figure 6B), does not generate ropes. Neither materials nor amorphous aggregates were formed by further C-terminal truncation mutants. Therefore, Ubx216-344 likely did not fail due to exposure of a second aggregation-prone region. We consequently conclude, within the resolution of our assays, that Ubx216-356 is a minimal materials-forming unit. As such, this region must include sequences permitting both fiber formation and fiber-fiber association. Curiously, none of the Ubx sequence features that resemble amyloidogenic or elastomeric proteins in the Nterminal 216 amino acids of the protein were required for materials formation, suggesting alternate mechanisms guide the assembly of materials from this portion of the Ubx sequence.


Figure 7. SEM of ropes generated from severely truncated Ubx variants. (A,B) Ropes composed of Ubx216-356 have uniform diameter and a striated surface, comparable to ropes composed of full-length Ubx. (C,D) On rare occasions, ropes generated from severely truncated Ubx variants have a rougher-appearing surface lacking striations, as shown here for a Ubx216-380 rope.

The decreased robustness of materials produced from Ubx216356 relative to full-length Ubx indicates that sequences outside this region impact materials assembly, even though these sequences are not absolutely required. This alteration in the quality of materials produced could be due to an alteration in the structure of the materials. However, SEM reveals that the surface of ropes composed of Ubx216-356 exhibits the aligned striations characteristic of Ubx ropes (Figure 7A). Occasionally, the surface of materials produced from severe truncations appears less smooth and uniform (Figure 7B), a trait never observed for ropes created with full-length Ubx. Thus, the appearance of Ubx216-356 materials is generally consistent with those composed of Ubx. Given the architecture of the Ubx216-356 materials generally appears intact, sequences outside this minimal domain must form additional interactions to enhance the robustness of the materials. One obvious mechanism would be for the Ubx sequence to contain a second region capable of forming materials and thus contributing additional stabilizing Ubx-Ubx interactions. To test this hypothesis, we generated C-terminal truncations in the context of full length Ubx (Figure 6B). Consistent with the existence of a second materials-forming domain, we could draw ropes using constructs with longer C-terminal truncations (past amino acid 356) when the N-terminal 215 amino acids were present. Indeed, Ubx1-243 generated ropes, although they were small and brittle. Using the Ubx1-243 C-terminal truncation mutant as a template, we then began deleting from the N-terminus to locate the N-terminal boundary of this second materials-forming region (Figure 6B). Ubx19-243, Ubx49-243, and N88-243 generated materials, but not Ubx139-243. Consequently, the N-terminal minimal materials-forming region in Ubx extends from amino acids 88 to 243, overlapping the C-terminal minimal materialsforming region by 28 amino acids, including the central alaninerich region (amino acids 221-234). This alanine-rich sequence is predicted to form an R-helix whose normal function in transcription activation relies on the potential to form secondary structure.22 Intriguingly, the N-terminal minimal materialsforming region (88-243) contains the disordered region, most of the GGX and GXXP motifs, and one alanine-rich region, and thus its sequence more closely resembles the sequences of


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known elastomeric, materials-forming proteins. Unfortunately, ropes generated from the N-terminal minimal materials-forming region appear even weaker than those produced by the Cterminal minimal materials-forming region. Consequently, we could not generate ropes from Ubx88-243 sufficiently long to transfer to a scanning electron microscope stub. Many traits are necessary in the minimal materials-forming regions. These sequences must be capable of forming fibers, the fibers must be capable of self-association, and regions inhibiting either type of assembly must be excluded or inhibited. Despite these multiple requirements, large sections of the Ubx sequence can removed without preventing materials formation: 63% of Ubx was removed to generate Ubx216-256, the C-terminal minimal materials-forming region, whereas 59% of the Ubx sequence was removed to generate Ubx88-243, the N-terminal minimal materials-forming region. However, these minimal regions only produce very short, brittle ropes. The entire Ubx sequence, including both minimal materials-forming domains as well as regions outside these domains, is required to generate the longer, more robust ropes analogous to ropes assembled from the full-length protein (Figure 6B, compare Ubx1-380 vs Ubx19-380 and Ubx1-356). Indeed, truncation from either the N-terminus or the C-terminus reduces the ability of Ubx to form ropes. Given the normal functions of Ubx (DNA binding, transcription activation and repression) can be localized to discrete domains within Ubx,19-24 it is interesting that robust materials assembly involves the entire sequence. Ubx Materials In Vivo. Given Ubx self-assembles so easily in Vitro, it is tempting to speculate that Ubx may also generate materials as part of its in ViVo function. However, extensive immunohistochemical analysis of the distribution and concentration of Ubx proteins in ViVo has not revealed extended Ubx oligomers within a Drosophila cell, even when the concentration of Ubx has been artificially elevated (e.g., refs 26 and 27). Furthermore, aggregates are not observed when overexpressing Ubx in E. coli. Since our data suggest Ubx requires exposure to an air-water interface to assemble in Vitro, it is unlikely that these microscale or macroscale materials form as part of the natural function of Ubx. Limited Ubx homomeric interactions do occur in ViVo upon binding a subset of Ubx-regulated enhancers that contain multiple(∼4to15)DNA-bindingsitesarrayedintandem.21,25Adjacent Ubx proteins appear to interact with one another to stabilize binding.25 Furthermore, Ubx proteins bound to multisite DNAs can interact with Ubx proteins bound to other DNA sites to create stem-loop DNA structures.25 At the molecular level, the protein interfaces used to form finite Ubx-Ubx interactions in ViVo may be similar to the interactions that drive nanoscale Ubx fiber assembly in Vitro. Therefore, while Ubx materials are probably not produced in ViVo, the protein-protein interactions that assemble Ubx materials may be biologically relevant.

Conclusions We were able to mimic the material architectures previously produced with elastomeric proteins as well as add novel structures by constructing materials from the Drosophila transcription factor Ubx, a protein that does not naturally form extended materials in ViVo. Within the Ubx sequence, we identified two regions capable of forming film and rope with an appearance comparable to those formed by full-length Ubx. However, generation of robust materials requires in-

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clusion of both minimal regions, and consequently the majority of the Ubx sequence. These materials are exceptionally interesting because (i) Ubx does not appear to generate these materials as part of its in ViVo function, (ii) the C-terminal minimal region lacks sequence features characteristic of other materials-forming proteins, (iii) the Nterminal materials-forming domain contains far fewer elastomeric motifs than proteins that form materials as part of their natural function, (iv) we were able to assemble materials using unusually gentle conditions, (v) Ubx ropes are surprisingly heat stable, and (vi) Ubx ropes are more extensible than collagen, spider dragline silk, and silkworm silk. These materials therefore provide a novel vantage point for exploring the structure-function-property relationships underlying organized protein self-association and materials production. The unusual properties of Ubx materials also offer several advantages for materials engineering relative to the elastomeric or amyloidogenic proteins frequently used to construct materials. The stability of the ubx gene in E. coli, combined with the ease of Ubx assembly, enables facile engineering, optimization, and functionalization of these materials. Full-length Ubx selfassembles within 2 h at protein concentrations under ambient conditions using 1-2 orders of magnitude less protein than required for structural protein-derived materials. These less stringent conditions are expected to greatly facilitate incorporation of chemically fragile ligands or aggregation-prone proteins via gene fusions, since many ligands and proteins would be inactivated by the harsh processing methods required to trigger assembly of most other recombinant protein-based materials.42,43 Finally, the self-adhesive properties of Ubx fibers permit hierarchical construction of complicated three-dimensional architectures. Acknowledgment. The authors thank the Robert A. Welch Foundation (C-576) and the Rice University Center for Biological and Environmental Nanotechnology (NSF EEX-0647452) for funding. We thank Robert White for the FP3.3.8 antibody, Ying Liu for providing expression constructs for Ubx19-380, Ubx49-380, Ubx88-380, Ubx139-380, Ubx174-380, and Ubx216380, Benjamin J. Greer for technical assistance, Jaimin Shah for assisting with protein purifications, and Cheng Peng for help with mechanical testing. Finally, we thank the members of the Bondos and Matthews laboratories for comments on the manuscript. Supporting Information Available. Table of conditions used to stimulate assembly of other protein systems, which cites refs 57-60, SDS-PAGE gel showing purity of Ubx and deletion mutants, and an experiment demonstrating changes in appearance of a Ubx solution upon film and sheet formation. This material is available free of charge via the Internet at http:// pubs.acs.org.

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BM801315V

The Drosophila Transcription Factor Ultrabithorax Self-Assembles into

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