Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Force and Scale Dependence of the Elasticity of Self-Assembled DNA Bottle Brushes
Rocha, Márcio Santos ; Storm, Ingeborg M. ; Bazoni, Raniella Falchetto ; Ramos, Ésio Bessa ; Hernandez-Garcia, Armando ; Cohen Stuart, Martien A. ; Leermakers, Frans ; Vries, Renko De - \ 2018
Macromolecules 51 (2018)1. - ISSN 0024-9297 - p. 204 - 212.
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.
Loss of bottlebrush stiffness due to free polymers
Storm, Ingeborg M. ; Kornreich, Micha ; Voets, Ilja K. ; Beck, Roy ; Vries, Renko de; Cohen Stuart, Martien A. ; Leermakers, Frans A.M. - \ 2016
Soft Matter 12 (2016)38. - ISSN 1744-683X - p. 8004 - 8014.

A recently introduced DNA-bottlebrush system, which is formed by the co-assembly of DNA with a genetically engineered cationic polymer-like protein, is subjected to osmotic stress conditions. We measured the inter-DNA distances by X-ray scattering. Our co-assembled DNA-bottlebrush system is one of the few bottlebrushes known to date that shows liquid crystalline behaviour. The alignment of the DNA bottlebrushes was expected to increase with imposed pressure, but interestingly this did not always happen. Molecularly detailed self-consistent field calculations targeted to complement the experiments, focused on the role of molecular crowding on the induced persistence length lp due to the side chains and the cross-sectional width D of the molecular bottlebrushes. Both the thickness as well as the backbone persistence length drop with increasing protein-polymer bulk concentrations and dramatic effects are found above the overlap threshold. The flexibilisation is more significant and therefore the bottlebrush aspect ratio, lp/D, decreases with protein-polymer concentration. This loss in aspect ratio is yet another argument why molecular bottlebrushes rarely order in anisotropic phases and may explain why bottlebrushes are excellent lubricants.

Nanofibrillar hydrogel scaffolds from recombinant protein-based polymers with integrin- and proteoglycan-binding domains
Wlodarczyk-Biegun, Gosia ; Werten, Marc W.T. ; Posadowska, Urszula ; Storm, Ingeborg M. ; Wolf, Frits A. de; Beucken, Jeroen J.J.P. van den; Leeuwenburgh, Sander C.G. ; Cohen Stuart, Martien ; Kamperman, Marleen - \ 2016
Journal of Biomedical Materials Research Part A 104 (2016)12. - ISSN 1549-3296 - p. 3082 - 3092.
KRSR cell-adhesive domain - Pichia pastoris - Protein-based polymers - Self-assembled hydrogel - Tissue engineering

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.

Co-assembled DNA-protein polymer bottlebrushes : main-chain stiffening & liquid crystallinity
Storm, I.M. - \ 2016
Wageningen University. Promotor(en): Martien Cohen Stuart; Frans Leermakers, co-promotor(en): Renko de Vries. - Wageningen : Wageningen University - ISBN 9789462577466 - 161 p.
polymers - liquid crystals - dna - proteins - polymeren - vloeibare kristallen - eiwitten

Bottlebrushes are macromolecules consisting of a backbone polymer onto which side chains are either physically or chemically grafted. Early theories suggested that attaching side chains to a (flexible) backbone molecule would induce the so-called main-chain stiffening effect. This newly formed bottlebrush molecule should therefore behave as a semi-flexible polymer rather than a flexible polymer. Due to this semi-flexible behaviour bottlebrushes should also be able to show liquid crystalline behaviour. However, there are very few examples of bottlebrush systems that are able to make liquid crystalline phases. In this thesis, we present a co-assembled bottlebrush system that consist of DNA as the backbone molecule and genetically engineered protein polymers as side chains. This co-assembled system is one of the few bottlebrush systems that actually does show liquid crystalline behaviour. This ability makes this bottlebrush system a perfect system to explain why it is so very difficult to make liquid crystalline phases with bottlebrushes. We have shown that attaching side chains will, at first, result in an effectively more flexible bottlebrush system. Only for systems with very densely packed and long side chains is the stiffness of the bottlebrush molecule increasing. Moreover, with osmotic stress experiments we have shown that the presence of free polymers also has a negative influence on the stiffness of bottlebrush molecules and hence this reduces the tendency for the system to form liquid crystals.

Physical and mechanical properties of thermosensitive xanthan/collagen-inspired protein composite hydrogels
Pham, Thao T.H. ; Snijkers, Frank ; Storm, Ingeborg M. ; Wolf, Frits A. De; Cohen Stuart, Martien A. ; Gucht, Jasper Van Der - \ 2016
International Journal of Polymeric Materials and Polymeric Biomaterials 65 (2016)3. - ISSN 0091-4037 - p. 125 - 133.
collagen-inspired protein - composite hydrogels - electrostatic interaction - strain-softening - thermosensitivity - Xanthan

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.

Monitoring protein capsid assembly with a conjugated polymer strain sensor
Cingil, E.H. ; Storm, I.M. ; Yorulmaz, Y. ; Brake, D.W. te; Vries, R.J. de; Cohen Stuart, M.A. ; Sprakel, J.H.B. - \ 2015
Journal of the American Chemical Society 137 (2015)31. - ISSN 0002-7863 - p. 9800 - 9803.
beta-phase formation - polyfluorene - poly(9,9-dioctylfluorene) - morphology - photophysics - copolymers - molecules - dynamics - length
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
Storm, I.M. ; Kornreich, M. ; Hernandez-Garcia, A. ; Voets, I.K. ; Beck, R. ; Cohen Stuart, M.A. ; Leermakers, F.A.M. ; Vries, R.J. de - \ 2015
The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical 119 (2015). - ISSN 1520-6106 - p. 4084 - 4092.
hyaluronan-aggrecan complexes - molecular bottle-brushes - neurofilament networks - boundary lubricants - articular-cartilage - lyotropic behavior - diblock copolymers - persistence length - mesomorphic state - click chemistry
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.
From micelles to fibers: balancing self-assembling and random coiling domains in pH-responsive silk-collagen-like protein-based polymers
Beun, L.H. ; Storm, I.M. ; Werten, M.W.T. ; Wolf, F.A. de; Cohen Stuart, M.A. ; Vries, R.J. de - \ 2014
Biomacromolecules 15 (2014)9. - ISSN 1525-7797 - p. 3349 - 3357.
elastin-like polypeptide - periodic polypeptides - pichia-pastoris - copolymers - hydrogels - sequence - nanoparticles - biosynthesis - secretion - networks
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.
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