Abstract
A central concept in molecular bioscience is how structure formation at different length scales is achieved. Here we use spider silk protein as a model to design new recombinant proteins that assemble into fibers. We made proteins with a three-block architecture with folded globular domains at each terminus of a truncated repetitive silk sequence. Aqueous solutions of these engineered proteins undergo liquid–liquid phase separation as an essential pre-assembly step before fibers can form by drawing in air. We show that two different forms of phase separation occur depending on solution conditions, but only one form leads to fiber assembly. Structural variants with one-block or two-block architectures do not lead to fibers. Fibers show strong adhesion to surfaces and self-fusing properties when placed into contact with each other. Our results show a link between protein architecture and phase separation behavior suggesting a general approach for understanding protein assembly from dilute solutions into functional structures.
Highlights
A central concept in molecular bioscience is how structure formation at different length scales is achieved
Variants were made with 2-block and 1-block architectures for eADF3, one with eADF3 attached to a single N-terminal cellulose-binding module (CBM) called CBM-eADF3, and the isolated eADF3 without added terminal-blocks
The formation of two different self-coacervated states, denoted as liquid-like coacervate (LLC) and solid-like coacervate (SLC), showed that the balance between surrounding conditions and protein architecture lead to different types of energy minima depending on solution conditions
Summary
A central concept in molecular bioscience is how structure formation at different length scales is achieved. We made proteins with a three-block architecture with folded globular domains at each terminus of a truncated repetitive silk sequence Aqueous solutions of these engineered proteins undergo liquid–liquid phase separation as an essential pre-assembly step before fibers can form by drawing in air. We show that two different forms of phase separation occur depending on solution conditions, but only one form leads to fiber assembly. The role of coacervates in the formation of biological materials such as squid beak has been described, in which again the preassembled state of proteins and low surface energy of the coacervate leads to efficient infiltration of a scaffold and subsequently to the formation of mechanically excellent structures[12,13]. Common to all these material assembly processes seems to be that the high polyelectrolyte concentration within coacervates is associated with an advantageous pre-assembly due to a molecular structuring within the coacervate[14,24]
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