Molecular design, self-assembly, and material properties of silk-like protein polymers

Research output: Thesisinternal PhD, WU



In this thesis we present the design and characterization of bio-inspired hybrid protein polymers. All polymers are composed of two distinct types of building blocks. The first type is a silk-inspired block that is pH-responsive and can fold and self-assemble into highly ordered structures. The basic structure of this building block is an octapeptide (GAGAGAGX), denoted SX. We include multiple repeats n of this octapeptide (SXn) in our protein polymers. The amino acid X is always an acidic or basic one. This way, the pH of the solvent determines the charge of this amino acid; in the charged state the silk-like blocks repel each other and the proteins are molecularly dissolved. When charge neutralized, the silk-like blocks become hydrophobic and can fold and stack. The second building block is very hydrophilic and acts as a random coil under a wide range of aqueous solvent conditions. The basic structure of this block is a 99 amino acids long sequence of mostly hydrophilic amino acids, that we included either as a single block or as multimers, denoted Cn. The combination of these two blocks in one molecule leads to a pH-responsive protein polymer that switches from hydrophilic to amphiphilic due to solvent pH. The amphiphilic nature of the neutralized protein polymer leads to microscopic phase separation.

All protein polymers were designed by genetic engineering and produced by genetically modified Pichia pastoris in a batch fermentation. A simple ammonium sulfate precipitation was sufficient for all types of proteins to acquire highly pure samples.

The types of hybrid protein polymers we produced and characterized differ in three aspects. Firstly, we designed different silk-like blocks in which the amino acid X had three varieties. We included the acidic amino acid glutamic acid (E) with a pKa ~ 4, to obtain a block that is charged at high pH values and neutral under acidic conditions. We also designed this silk-like block with basic amino acids lysine (K) or histidine (H). These blocks are positively charged under acidic conditions, while being neutral at higher pH. Lysine with a pKa ~ 10 remains charged under slightly alkaline conditions, and is only neutralized at rather extreme pH values. The pKa of histidine (~ 6) means this is the only pH-responsive amino acid that’s almost completely neutral under physiological conditions (a pH of 7.4). This makes histidine the most interesting residue from a biomedical point of view.

The second variety in design is the relative sizes of the two blocks. For any amphiphile, the relative sizes of the two blocks determine the structure that is formed upon self-assembly. The size of the silk-like block was chosen to be 8, 16, 24 or 48 repeats of the octapeptide SXN. The random coil block was included as monomer C1, dimer C2 or tetramer C4.

Our third variation in molecular design is the order of the two building blocks. We constructed two diblock structured protein polymers: C1SH48 and C2SH48. All other protein polymers had a triblock structure. We constructed C2SXNC2 protein polymers, where the self-assembling silk-like domain was the central block, and SX24C4SX24 protein polymers, with telechelic end blocks. All types of protein polymers that we studied are presented in Table 6.1.

Table 6.1: Overview of different protein polymers studied in this thesis.


(Ch. 2)

Varying Silk-Like Block

(Ch. 3 & 4)

Varying Random Coil Block

(Ch. 5)













In chapter 2 we report on the self-assembly behavior of triblock structured telechelic protein polymers SX24C4SX24. We analyzed the pH-dependent self-assembly into fibrillar structures of three different protein polymers. These proteins only differ in the amino acid X in the silk-like block. We found that all proteins self-assemble into fibrils under solvent conditions at which the amino acid X is uncharged. This self-assembly is completely reversible; changing the solvent pH to a value at which the amino acid X is fully charged, leads to immediate disassembly of the fibrils. The secondary structures of the fibrils are comparable, and are a combination of a random coil corona and a crystalline folded and self-assembled core. The self-assembly process is a pseudo-first order one. Initial fast (heterogeneous) nucleation is followed by elongation of existing fibrils, without the formation of new fibrils. These kinetics lead to monodisperse samples of fibrils. Existing fibrils have at least one living end: addition of new proteins in solution leads to further growth of these fibrils.  

In chapter 3 we use super resolution fluorescence microscopy and atomic force microscopy to analyze the self-assembly mechanism of the protein polymer C2SH48C2. Surprisingly, we found that self-assembly of these fibrils is an asymmetric process. The fibrils grow in only one direction with one living end, although the protein polymer that is the building block of these fibrils is highly symmetric. We therefore conclude that nucleation is a heterogeneous process. We observed that once a protein molecule is part of a fibril, it is kinetically trapped. In a timeframe of 3 days, we don’t observe exchange of protein molecules inside fibrils, with proteins in solution. The interactions of uncharged folded protein polymers inside a fibril are simply too strong to overcome to be released into solution. We also report that self-assembly of these fibrils is a process that involves continuous nucleation; elongation of existing fibrils is accompanied by the genesis of new ones. This leads to samples that contain fibrils with a wide variety of sizes, quite different from the populations found for the protein polymers with the inverted block sequence that we presented in chapter 2.

In chapter 4 and 5 we present our findings on several protein polymers in which we varied the relative sizes of the silk-like block and of the random coil blocks. In chapter 4 we present the characterization of protein polymers that have differently sized silk-like domains. We studied 4 protein polymers with the general structure C2SHnC2; the series consisted of n = 8, 16, 24 or 48. The two smallest protein polymers form micelles when charge neutralized (pH 8). The two largest protein polymers form fibrils under these conditions. At low pH, when the silk-like block is highly charged, this block behaves as (extended) random coil, according to circular dichroism measurements. This behavior is consistent for all block sizes that we studied. In the self-assembled state, there is a distinct difference in secondary structure of the micelles and the fibrils. The silk-like core of the micelles has a secondary structure that differs only slightly from the structure in the charged state. It merely acts as a collapsed coil. The secondary structures of the fibril forming protein polymers are very different in neutralized state. Their structures are mutually nearly identical, similar to that of a betaroll.

We observed that the size of the silk-like block has a strong effect on the kinetics of self-assembly. The largest protein polymer C2SH48C2 self-assembles into fibrils at a rate that is over a decade faster than the protein polymer with the smaller silk-like block C2SH24C2. Both fibril forming protein polymers can form hydrogels. There is however a great difference in rigidity of the gels at similar concentrations. The gel that consists of fibrils of C2SH48C2 is a decade stiffer than the one consisting of C2SH24C2. This stronger fibril-fibril interaction due to the more exposed silk-like core of C2SH48C2 clearly has a strong effect on macroscopic gel properties. We used partial enzymatic degradation of the random coil block to determine the influence of decreasing the hydrophilic block on self-assembly behavior. Both micelle forming protein polymers are able to form fibrils after up to 80% of the random coil blocks has been cleaved off. This shows the intrinsic capacity of the silk-like block to form fibrils even at a size as small as 64 amino acids. The fibrils of C2SH24C2 show an increase in interaction after partial cleavage of the random coil block. Individual fibrils start to associate laterally. This is a strong indication that fibrils with smaller random coil blocks can form more rigid hydrogels, based on their increased core-core interaction.

Based on these findings, we designed two new protein polymers with smaller random coil blocks: C1SH48 and C2SH48. With these protein polymers we systematically probe the role of a more exposed silk-like core in gel properties, presented in chapter 5. Both proteins self-assemble into fibrils when neutralized. Fibrils of C1SH48 differ from those of  C2SH48 and C2SH48C2 as they start to laterally associate. Surprisingly, the rates at which self-assembling fibrils are formed, are identical for these protein polymers, and also equal to the rate of C2SH48C2­. Apparently, for these sizes of the blocks, the size of the silk-like block is what determines the rate of self-assembly. These two protein polymers attain secondary structures that are very similar to that of C2SH48C2. When looking at macroscopic properties of hydrogels formed by these protein polymers, we do observe a very clear difference. Both C2SH48C2 and C2SH48 form fibril based hydrogels that act as gels with very few (weak) crosslinks. These two gels show similar scaling behavior of modulus with concentration (exponents of 1.52 and 1.66). The attractive interaction of fibrils of C1SH48 leads to a different type of gel. The modulus of this hydrogel scales strongly with concentration (exponent of 2.8), typical for a (physically) cross-linked gel. The latter gels can have much greater moduli than gels of C2SH48C2 and C2SH48, but are also slightly more brittle. The porosity of the gels (an important parameter for biomedical applications) increases when decreasing the size of the random coil domain. However, for this series of protein polymers, the porosity is in the order of 10-100 nm, which makes these gels not suitable for using them to grow 3D cell cultures.


Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • Cohen Stuart, Martien, Promotor
  • de Vries, Renko, Co-promotor
Award date13 Mar 2015
Place of PublicationWageningen
Print ISBNs9789462572331
Publication statusPublished - 2015


  • protein engineering
  • protein
  • self assembly
  • design
  • polymerization
  • collagen


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