Work

Negative stain TEM imaging of native spider silk protein superstructures

Year: 2025

Type: article

Abstract: • A protocol to stain native dragline spider silk proteins with ammonium molybdate is developed. • Several morphologies were observed with TEM: fibers, large superstructures, and small micelles. • Particles measured in ImageJ were grouped into two populations averaging 50 nm and 300 nm. • The 50 nm size population was more prevalent at higher urea concentrations. • Similar distributions of particles were observed with DLS. Native Latrodectus hesperus (black widow) major ampullate spider silk proteins were imaged using negative stain transmission electron microscopy (NS-TEM) by isolating the silk protein hydrogel directly from the organism and solubilizing in urea. Heterogeneous micelle-like structures averaging 300 nm, similar to those imaged previously with CryoEM, were observed when stained with ammonium molybdate. A second smaller population averaging 50 nm was observed as well as large fibrils, highlighting the heterogeneous nature of the silk gland. The population of smaller silk protein micelles was enhanced at higher urea concentrations (5–8 M). This was further supported by dynamic light scattering (DLS), where two populations were observed at low urea concentrations while one small population dominated at high urea concentrations. The approach presented here provides a cost-effective route to imaging silk protein superstructures with conventional NS-TEM methods and may be applicable to other soft nanoparticle systems.

Source: Ultramicroscopy

DOI: 10.1016/j.ultramic.2025.114124

Authors: Hannah R. Johnson, Legend Foster, Anikin Rae Domingo, Gregory P. Holland

Institution: San Diego State University

Language: English

Topic: Silk-based biomaterials and applications

Subfield: Biomaterials

Field: Materials Science

Domain: Physical Sciences

Open Access status: hybrid

Estimated APC paid: $3,760

Funder: Air Force Office of Scientific Research