Phosphate- and pH-dependent self-assembly of recombinant spider silk proteins

Title data

Trossmann, Vanessa T. ; Hovanová, Veronika ; Schiller, Tim ; Humenik, Martin ; Sedlák, Erik ; Scheibel, Thomas:
Phosphate- and pH-dependent self-assembly of recombinant spider silk proteins.
In: Protein Science. Vol. 35 (2026) Issue 5 . - e70554.
ISSN 1469-896X
DOI: https://doi.org/10.1002/pro.70554

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Abstract in another language

The process of molecular self-assembly is an omnipresent mechanism in nature to generate a variety of efficient and functional hierarchical architectures, and inspires tailored material design and development. Thereby, self-assembly is based on a controlled interplay and association of monomers into highly ordered structures triggered by different non-covalent interactions. However, in the context of structural protein self-assembly, this association process could be influenced by the underlying amino acid sequence as well as external triggers including pH value, protein concentration, or ionic composition. Thus, understanding their impact on protein conformation and assembly is indispensable for controlled protein processing and functional materials' engineering. Here, we analyzed the self-assembly behavior of the intrinsically unstructured, recombinant spider silk proteins eADF4(Ω16) and eADF4(C16), which only differ in one amino acid residue in their repetitive module (glutamine and glutamic acid, respectively), depending on the concentration of kosmotropic potassium phosphate (KPi) and the pH value. The low protein charge in eADF4(Ω16) at neutral pH led to a compacted protein conformation and a significantly increased sensitivity to phosphate resulting in faster assembly kinetics of nanofibrils and precipitation of particles at lower KPi concentrations. In contrast, the presence of glutamic acid residues in eADF4(C16) enhanced the solubility and stability of protein monomers above physiological pH but led to an enhanced assembly/aggregation along with decreasing pH-values. Interestingly, deprotonation of tyrosine residues at pH 10 introduced negative charges resulting in decreased hydrophobic interactions and thus decelerated restructuring and assembly of eADF4(Ω16). Our results enabled the identification of Pi- and pH-dependent conformation and assembly models of eADF4-based spider silk proteins allowing controllable processing into fibrils, particles, or hydrogels for specific applications.