N-Terminal domain of Bombyx mori fibroin mediates the assembly of silk in response to pH decrease

Area of Science:

• Biochemistry
• Structural Biology
• Materials Science

Background:

• Silk fibroin, the primary component of silk fiber, is a large protein crucial for silk's mechanical properties.
• The fibroin heavy chain contains hydrophilic domains and repetitive Gly-Ala regions, influencing its assembly and structure.
• Understanding the self-assembly mechanisms of silk fibroin is key to its biomaterial applications.

Purpose of the Study:

• To elucidate the structural and functional properties of the silk fibroin N-terminal domain (FibNT).
• To investigate the pH-responsive conformational changes and self-assembly behavior of FibNT.
• To identify the molecular determinants governing FibNT's role in silk fibroin assembly.

Main Methods:

• X-ray crystallography was used to determine the structure of FibNT at pH 4.7.
• Dynamic light scattering and electron microscopy were employed to study FibNT oligomerization and morphology.
• Mutagenesis analysis identified key residues involved in pH-dependent conformational transitions.

Main Results:

• The crystal structure of FibNT revealed a unique double-layered anti-parallel β-sheet structure.
• FibNT undergoes a pH-responsive transition from a random coil to β-sheets around pH 6.0.
• FibNT forms micelle-like oligomers upon decreasing pH, consistent with the silk fibroin micelle assembly model.
• Conserved acidic residues, Glu56 and Asp100, were found to inhibit premature β-sheet formation at neutral pH.

Conclusions:

• The silk fibroin N-terminal domain (FibNT) acts as a pH-responsive self-assembly module.
• FibNT's ability to prevent premature β-sheet formation at neutral pH is critical for controlled silk assembly.
• This pH-dependent mechanism facilitates fibroin assembly as it moves through the posterior to anterior silk gland.