Ingeborg M. Storm

From micelles to fibers: balancing self-assembling and random coiling domains in pH-responsive silk-collagen-like protein-based polymers

We study the self-assembly of genetically engineered protein-based triblock copolymers consisting of a central pH-responsive silk-like middle block (SHn, where SH is a silk-like octapeptide, (GA)3GH and n is the number of repeats) flanked by hydrophilic random coil outer blocks (C2). Our previous work has already shown that triblocks with very long midblocks (n = 48) self-assemble into long, stiff protein filaments at pH values where the middle blocks are uncharged. Here we investigate the self-assembly behavior of the triblock copolymers for a range of midblock lengths, n = 8, 16, 24, 48. Upon charge neutralization of SHn by adjusting the pH, we find that C2SH8C2 and C2SH16C2 form spherical micelles, whereas both C2SH24C2 and C2SH48C2 form protein filaments with a characteristic beta-roll secondary structure of the silk midblocks. Hydrogels formed by C2SH48C2 are much stronger and form much faster than those formed by C2SH24C2. Enzymatic digestion of much of the hydrophilic outer blocks is used to show that with much of the hydrophilic outer blocks removed, all silk-midblocks are capable of self-assembling into stiff protein filaments. In that case, reduction of the steric repulsion by the hydrophilic outer blocks also leads to extensive fiber bundling. Our results highlight the opposing roles of the hydrophilic outer blocks and central silk-like midblocks in driving protein filament formation. They provide crucial information for future designs of triblock protein-based polymers that form stiff filaments with controlled bundling, that could mimick properties of collagen in the extracellular matrix.

Monitoring protein capsid assembly with a conjugated polymer strain sensor

Semiconducting polymers owe their optoelectronic properties to the delocalized electronic structure along their conjugated backbone. Their spectral features are therefore uniquely sensitive to the conformation of the polymer, where mechanical stretching of the chain leads to distinct vibronic shifts. Here we demonstrate how the optomechanical response of conjugated polyelectrolytes can be used to detect their encapsulation in a protein capsid. Coating of the sensor polymers by recombinant coat proteins induces their stretching due to steric hindrance between the proteins. The resulting mechanical planarizations lead to pronounced shifts in the vibronic spectra, from which the process of capsid formation can be directly quantified. These results show how the coupling between vibronic states and mechanical stresses inherent to conjugated polymers can be used to noninvasively measure strains at the nanoscale.

Liquid crystals of self-assembled DNA bottlebrushes

Early theories for bottlebrush polymers have suggested that the so-called main-chain stiffening effect caused by the presence of a dense corona of side chains along a central main chain should lead to an increased ratio of effective persistence length (lp,eff) over the effective thickness (Deff) and, hence, ultimately to lyotropic liquid crystalline behavior. More recent theories and simulations suggest that lp,eff ∼ Deff, such that no liquid crystalline behavior is induced by bottlebrushes. In this paper we investigate experimentally how lyotropic liquid crystalline behavior of a semiflexible polymer is affected by a dense coating of side chains. We use semiflexible DNA as the main chain. A genetically engineered diblock protein polymer C4K12 is used to physically adsorb long side chains on the DNA. The C4K12 protein polymer consists of a positively charged binding block (12 lysines, K12) and a hydrophilic random coil block of 400 amino acids (C4). From light scattering we find that, at low ionic strength (10 mM Tris-HCl), the thickness of the self-assembled DNA bottlebrushes is on the order of 30 nm and the effective grafting density is 1 side chain per 2.7 nm of DNA main chain. We find these self-assembled DNA bottlebrushes form birefringent lyotropic liquid crystalline phases at DNA concentrations as low as 8 mg/mL, roughly 1 order of magnitude lower than for bare DNA. Using small-angle X-ray scattering (SAXS) we show that, at DNA concentrations of 12 mg/mL, there is a transition to a hexagonal phase. We also show that, while the effective persistence length increases due to the bottlebrush coating, the effective thickness of the bottlebrush increases even more, such that in our case the bottlebrush coating reduces the effective aspect ratio of the DNA. This is in agreement with theoretical estimates that show that, in most cases of practical interest, a bottlebrush coating will lead to a decrease of the effective aspect ratio, whereas, only for bottlebrushes with extremely long side chains at very high grafting densities, a bottlebrush coating may be expected to lead to an increase of the effective aspect ratio.

Nanofibrillar hydrogel scaffolds from recombinant protein‐based polymers with integrin‐ and proteoglycan‐binding domains

Abstract This study describes the design, production, and testing of functionalized variants of a recombinant protein‐based polymer that forms nanofibrillar hydrogels with self‐healing properties. With a view to bone tissue engineering applications, we equipped these variants with N‐terminal extensions containing either (1) integrin‐binding (RGD) or (2) less commonly studied proteoglycan‐binding (KRSR) cell‐adhesive motifs. The polymers were efficiently produced as secreted proteins using the yeast Pichia pastoris and were essentially monodisperse. The pH‐responsive protein‐based polymers are soluble at low pH and self‐assemble into supramolecular fibrils and hydrogels at physiological pH. By mixing functionalized and nonfunctionalized proteins in different ratios, and adjusting pH, hydrogel scaffolds with the same protein concentration but varying content of the two types of cell‐adhesive motifs were readily obtained. The scaffolds were used for the two‐dimensional culture of MG‐63 osteoblastic cells. RGD domains had a slightly stronger effect than KRSR domains on adhesion, activity, and spreading. However, scaffolds featuring both functional domains revealed a clear synergistic effect on cell metabolic activity and spreading, and provided the highest final degree of cell confluency. The mixed functionalized hydrogels presented here thus allowed to tailor the osteoblastic cell response, offering prospects for their further development as scaffolds for bone regeneration.

Recombinant Protein Polymers for Colloidal Stabilization and Improvement of Cellular Uptake of Diamond Nanosensors

Fluorescent nanodiamonds are gaining increasing attention as fluorescent labels in biology in view of the fact that they are essentially nontoxic, do not bleach, and can be used as nanoscale sensors for various physical and chemical properties. To fully realize the nanosensing potential of nanodiamonds in biological applications, two problems need to be addressed: their limited colloidal stability, especially in the presence of salts, and their limited ability to be taken up by cells. We show that the physical adsorption of a suitably designed recombinant polypeptide can address both the colloidal stability problem and the problem of the limited uptake of nanodiamonds by cells in a very straightforward way, while preserving both their spectroscopic properties and their excellent biocompatibility.

Force and Scale Dependence of the Elasticity of Self-Assembled DNA Bottle Brushes

As a model system to study the elasticity of bottle-brush polymers, we here introduce self-assembled DNA bottle brushes, consisting of a DNA main chain that can be very long and still of precisely defined length, and precisely monodisperse polypeptide side chains that are physically bound to the DNA main chains. Polypeptide side chains have a diblock architecture, where one block is a small archaeal nucleoid protein Sso7d that strongly binds to DNA. The other block is a net neutral, hydrophilic random coil polypeptide with a length of exactly 798 amino acids. Light scattering shows that for saturated brushes the grafting density is one side chain per 5.6 nm of DNA main chain. According to small-angle X-ray scattering, the brush diameter is D = 17 nm. By analyzing configurations of adsorbed DNA bottle brushes using AFM, we find that the effective persistence of the saturated DNA bottle brushes is Peff = 95 nm, but from force–extension curves of single DNA bottle brushes measured using optical tweezers we find Peff = 15 nm. The latter is equal to the value expected for DNA coated by the Sso7d binding block alone. The apparent discrepancy between the two measurements is rationalized in terms of the scale dependence of the bottle-brush elasticity using theory previously developed to analyze the scale-dependent electrostatic stiffening of DNA at low ionic strengths.

Physical and mechanical properties of thermosensitive xanthan/collagen-inspired protein composite hydrogels

ABSTRACT Functionalization of xanthan hydrogels is of interest for biomaterial applications. The authors report characterization of electrostatic complexation of xanthan with a recombinant collagen-inspired triblock protein polymer. This polymer has one charged polylysine end-block that can bind to xanthan by electrostatic interactions, and another end-block that can self-assemble into thermosensitive collagen-like triple helices; the end-blocks are connected by a neutral, hydrophilic, mostly inert random coil. The protein modifies the xanthan/protein composite hydrogels in three ways: (a) a significant increase in storage modulus, (b) thermosensitivity, and (c) a two-step strain softening in nonlinear rheology. GRAPHICAL ABSTRACT