Phase transitions as intermediate steps in the formation of molecularly engineered protein fibers

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.

Self-Coacervation of a Silk-Like Protein and Its Use As an Adhesive for Cellulosic Materials

“… 11 Consequently, new types of biological materials are expected. We noted previously that a block-architecture in which a spidroin repetitive sequence is flanked in both N and C termini by folded domains leads to a self-coacervating phase behavior 12 ( Figures 1 A and S1–S4 ). The general architecture was important for coacervation, and the same behavior was achieved even with other unrelated proteins used as terminal domains.…”

“…To make a distinction to previously described phase separation that was achieved by adding potassium phosphate, we name the system under study here LLC (liquid-like coacervate (or condensate)). 12 The LLCs form spontaneously when protein concentration is a high and ionic strength is low. The LLC-containing phase remained stable at even 30% protein concentration.…”

Biomimetic Composites with Enhanced Toughening Using Silk Inspired Triblock Proteins and Aligned Nanocellulose Reinforcements

“…Cloning, expression, and protein purification Cloning, expression, and protein purification were carried out as described earlier (18,20). Briefly, DNA sequences encoding bacterial family three CBMs (CBM3) from Ruminiclostridium thermocellum (Protein Data Bank accession number 1NBC) (21), DNA sequence encoding a 499-amino acid stretch of the A. diadematus dragline spidroin (ADF3), and a 12-time repeat of residues 325 to 368 in ADF3 (eADF3) (11,35,36) were synthesized and codon-optimized by GeneArt Gene Synthesis (Thermo Fisher Scientific) for expression in Escherichia coli.…”

Analyzing the weak dimerization of a cellulose binding module by sedimentation velocity experiments

“…As the K D for the binding of CBM CipA is at around 0.6 μM (13–15), which is around two orders of magnitude lower we conclude that its dimerization has a negligible effect on any function involving cellulose binding. However, in other uses of the CBM such as using then as part of molecular adhesives, much higher concentrations were used reaching over 2 mM (11,17). At these concentrations the majority of protein molecules are in the dimer form, unless the K D is affected when the CBM CipA is part of a fusion protein.…”

“…Because of its wide use and because any multimerization behavior possibly can influence the use of the protein in the applications, we decided to investigate the multimerization characteristics of CBM CipA in more detail. Furthermore, a possibility of dimerization was indicated in our previous work using CBM CipA as a fusion partner in silk-material forming proteins (11,17,18). For CBM CipA , no detailed information of dimerization is available, but the formation of dimers has been reported for the closely related CBD Cex from the enzyme Cex, a β-1,4-glycanase from C.fimi (19).…”