Negative stain TEM imaging of native spider silk protein superstructures

Area of Science:

• Biomaterials Science
• Structural Biology
• Materials Science

Background:

• Spider silk proteins, particularly from the major ampullate gland of Latrodectus hesperus (black widow), are known for their remarkable mechanical properties.
• Understanding the self-assembly and structural organization of these proteins is crucial for biomimetic material design.
• Previous imaging techniques like CryoEM have provided insights into silk protein structures.

Purpose of the Study:

• To investigate the structural characteristics of black widow spider silk proteins using negative stain transmission electron microscopy (NS-TEM).
• To explore the effect of urea concentration on the self-assembly of silk proteins.
• To establish a cost-effective method for imaging silk protein superstructures.

Main Methods:

• Isolation of silk protein hydrogel directly from native Latrodectus hesperus.
• Solubilization of silk proteins in urea and imaging via NS-TEM with ammonium molybdate staining.
• Dynamic Light Scattering (DLS) analysis to corroborate structural observations.

Main Results:

• Observation of heterogeneous micelle-like structures averaging 300 nm and a smaller population averaging 50 nm, alongside large fibrils.
• Confirmation of structural heterogeneity within the silk gland.
• Enhancement of smaller silk protein micelle populations at higher urea concentrations (5-8 M).
• DLS data showed two populations at low urea concentrations, with a single small population dominating at high concentrations.

Conclusions:

• Native black widow spider silk proteins form heterogeneous micelle-like structures and fibrils.
• Urea concentration significantly influences the observed silk protein superstructure populations.
• NS-TEM offers a cost-effective alternative for imaging silk protein superstructures, applicable to other soft nanoparticle systems.