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

Beun LH, Storm IM, Werten MW, de Wolf FA, Cohen Stuart MA, de Vries R - Biomacromolecules (2014)

Bottom Line: Hydrogels formed by C2S(H)48C2 are much stronger and form much faster than those formed by C2S(H)24C2.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Physical Chemistry and Colloid Science, Wageningen University , Dreijenplein 6, NL-6703 HB Wageningen, The Netherlands.

ABSTRACT
We study the self-assembly of genetically engineered protein-based triblock copolymers consisting of a central pH-responsive silk-like middle block (S(H)n, where S(H) 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 S(H)n by adjusting the pH, we find that C2S(H)8C2 and C2S(H)16C2 form spherical micelles, whereas both C2S(H)24C2 and C2S(H)48C2 form protein filaments with a characteristic beta-roll secondary structure of the silk midblocks. Hydrogels formed by C2S(H)48C2 are much stronger and form much faster than those formed by C2S(H)24C2. 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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Storage modulus in timeof 25 g/L solutions of C2SH24C2 and C2SH48C2 directlyafter quenching to pH 8 in 50 mM phosphatebuffer at 298 K.
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fig7: Storage modulus in timeof 25 g/L solutions of C2SH24C2 and C2SH48C2 directlyafter quenching to pH 8 in 50 mM phosphatebuffer at 298 K.

Mentions: C2SH48C2 is already known toform hydrogels at neutral or higher pH,32 at weight concentrations exceeding 10 g/L. Herewe have shown that the C2SH24C2 protein also self-assembles into protein filaments, and thatafter prolonged incubation, essentially all protein is incorporatedin protein filaments. Next, we follow the gelation of 25 g/L solutionsof both proteins by online rheometry, as a function of the incubationtime at pH 8, for a time period of up to 2 days. Figure 7 shows the development of the storage modulus of both solutionsin time. There are two distinct differences between the curves forthe two proteins. First, gelation of C2SH24C2 is very much slower than that of C2SH48C2. The graph shows a lag timeof several hours before the storage modulus starts increasing, whileC2SH48C2 starts gellingvirtually instantaneously. This observation is in line with our findingswith Time Resolved AFM of much slower filament growth rates. Apparently,filaments of C2SH24C2 growso slowly that it takes a significant time to reach the overlap concentration,while this transition point is reached much faster for the case ofC2SH48C2. Second, thelimiting value of the storage modulus (after 48 h of incubation timeat pH 8) differs by almost an order of magnitude. Since it appearsthat all protein is eventually incorporated into protein filaments,at identical weight concentrations, we anticipate that the total lengthof protein filament should be roughly equal, and the difference observedmust be due to differences in either the length or structural organizationof the fibers in the network structure. For dilute samples we haveobserved that final filament lengths are comparable for the two proteins.Assuming that this also holds for more concentrated samples, a possiblecause could be a difference in filament–filament interactions,that lead to a different structural organization of the fibers inthe network structure. The C2SH48C2 fibers have twice the exposed histidine rich (hydrogenbonding and aromatic character) surface area as compared to C2SH24C2 fibers, and this mightleads to a stronger attractive force between fibers. The differencein size of the tightly packed silk-like domain in the protein filamentsmay result in a difference in stiffness of the filaments. This mightcontribute to the difference in gel properties as well. If these hypothesesare true, a further increase of the silk-like domain, or a decreaseof the random coiling domain (facilitating contact between the silk-likedomains of neighboring filaments) should lead to stronger hydrogels,at even lower concentrations than those we observe here for C2SH48C2.


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

Beun LH, Storm IM, Werten MW, de Wolf FA, Cohen Stuart MA, de Vries R - Biomacromolecules (2014)

Storage modulus in timeof 25 g/L solutions of C2SH24C2 and C2SH48C2 directlyafter quenching to pH 8 in 50 mM phosphatebuffer at 298 K.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260859&req=5

fig7: Storage modulus in timeof 25 g/L solutions of C2SH24C2 and C2SH48C2 directlyafter quenching to pH 8 in 50 mM phosphatebuffer at 298 K.
Mentions: C2SH48C2 is already known toform hydrogels at neutral or higher pH,32 at weight concentrations exceeding 10 g/L. Herewe have shown that the C2SH24C2 protein also self-assembles into protein filaments, and thatafter prolonged incubation, essentially all protein is incorporatedin protein filaments. Next, we follow the gelation of 25 g/L solutionsof both proteins by online rheometry, as a function of the incubationtime at pH 8, for a time period of up to 2 days. Figure 7 shows the development of the storage modulus of both solutionsin time. There are two distinct differences between the curves forthe two proteins. First, gelation of C2SH24C2 is very much slower than that of C2SH48C2. The graph shows a lag timeof several hours before the storage modulus starts increasing, whileC2SH48C2 starts gellingvirtually instantaneously. This observation is in line with our findingswith Time Resolved AFM of much slower filament growth rates. Apparently,filaments of C2SH24C2 growso slowly that it takes a significant time to reach the overlap concentration,while this transition point is reached much faster for the case ofC2SH48C2. Second, thelimiting value of the storage modulus (after 48 h of incubation timeat pH 8) differs by almost an order of magnitude. Since it appearsthat all protein is eventually incorporated into protein filaments,at identical weight concentrations, we anticipate that the total lengthof protein filament should be roughly equal, and the difference observedmust be due to differences in either the length or structural organizationof the fibers in the network structure. For dilute samples we haveobserved that final filament lengths are comparable for the two proteins.Assuming that this also holds for more concentrated samples, a possiblecause could be a difference in filament–filament interactions,that lead to a different structural organization of the fibers inthe network structure. The C2SH48C2 fibers have twice the exposed histidine rich (hydrogenbonding and aromatic character) surface area as compared to C2SH24C2 fibers, and this mightleads to a stronger attractive force between fibers. The differencein size of the tightly packed silk-like domain in the protein filamentsmay result in a difference in stiffness of the filaments. This mightcontribute to the difference in gel properties as well. If these hypothesesare true, a further increase of the silk-like domain, or a decreaseof the random coiling domain (facilitating contact between the silk-likedomains of neighboring filaments) should lead to stronger hydrogels,at even lower concentrations than those we observe here for C2SH48C2.

Bottom Line: Hydrogels formed by C2S(H)48C2 are much stronger and form much faster than those formed by C2S(H)24C2.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Physical Chemistry and Colloid Science, Wageningen University , Dreijenplein 6, NL-6703 HB Wageningen, The Netherlands.

ABSTRACT
We study the self-assembly of genetically engineered protein-based triblock copolymers consisting of a central pH-responsive silk-like middle block (S(H)n, where S(H) 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 S(H)n by adjusting the pH, we find that C2S(H)8C2 and C2S(H)16C2 form spherical micelles, whereas both C2S(H)24C2 and C2S(H)48C2 form protein filaments with a characteristic beta-roll secondary structure of the silk midblocks. Hydrogels formed by C2S(H)48C2 are much stronger and form much faster than those formed by C2S(H)24C2. 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.

Show MeSH
Related in: MedlinePlus