Phase separation drives the folding of recombinant collagen

Area of Science:

• Biomaterials Science
• Protein Engineering
• Structural Biology

Background:

• Recombinant collagens offer sustainable alternatives to natural sources but face challenges in achieving proper folding.
• Controlling collagen folding is essential for replicating the mechanical properties of natural materials.
• Willow sawfly collagen offers a simpler, non-hydroxylated alternative to mammalian collagens.

Purpose of the Study:

• To investigate methods for controlling the folding and assembly of recombinant collagens.
• To explore the potential of phase separation in inducing collagen triple helix formation.
• To establish a universal mechanism for enhancing recombinant collagen folding.

Main Methods:

• Circular Dichroism (CD) spectroscopy to analyze protein structure.
• Analytical ultracentrifugation to study protein assembly and concentration dependence.
• Microscopy to visualize phase separation induced by freezing and crowding agents.

Main Results:

• Recombinant willow sawfly collagen self-assembles into triple helices in a concentration-dependent manner.
• Freezing and crowding agents overcome the folding concentration threshold by inducing phase separation.
• Phase separation increases local protein concentration, driving collagen folding nucleation.
• Freezing also promotes folding of human collagen fragments and accelerates bovine collagen folding.

Conclusions:

• Phase separation is a viable strategy to induce and control recombinant collagen folding.
• This approach offers a universal method for enhancing collagen assembly without genetic modification or crosslinking.
• The findings provide a new tool for producing functional recombinant collagens for biomaterial applications.