# Structural and dynamic similarities of nanofibrils and microparticles of engineered spider silk proteins probed by solid‐state NMR spectroscopy

## TL;DR

This study uses NMR to explore the structure and dynamics of engineered spider silk proteins, revealing similarities between different silk morphologies.

## Contribution

The study provides novel structural insights into engineered spider silk proteins using solid-state NMR, revealing atomic-level similarities across different silk morphologies.

## Key findings

- Nanofibrils and microparticles of eADF4(C16) show high structural and dynamic similarities at the atomic level.
- Tyrosine sidechains are rigidified, suggesting π–π-stacking interactions, and glutamic acid residues are deprotonated.
- Proline residues exhibit trans- and cis-conformations, potentially controlling β-sheet formation during self-assembly.

## Abstract

Spider silks are proteinaceous fiber materials inspiring material design in various technical and biomedical fields due to their exceptional toughness, which exceeds that of most natural and artificial fibers. Solid‐state nuclear magnetic resonance (NMR) spectroscopy has been used herein to obtain insights into the structure and dynamics of 13C/15N isotope‐labeled nanofibrils and microparticles made of the recombinantly produced, engineered spider silk protein eADF4(C16), for which structural information was still lacking. Although these two β‐sheet‐rich morphologies differ substantially in their microscopic appearance (nanofibrils vs. microparticles), the solid‐state NMR spectra reveal high structural and dynamic similarities at the atomic level. For both morphologies, it was found that the rigid alanine stretch in the eADF4 sequence forms a mixture of rectangular and staggered β‐sheets extending to the flanking serine residues. In addition, our data reveal that the tyrosine sidechains are rigidified, which suggests their engagement in π–π‐stacking interactions. All of the glutamic acid residues were found to be deprotonated, which implies their localization on the outside of the fibril, where their negative charge can be compensated. Trans‐ as well as cis‐conformations were observed for proline residues, which suggests that they might further control the formation and extension of the poly‐alanine β‐sheet region during the self‐assembly process. The gained understanding of structure, dynamics, and assembly of the engineered spider silk protein eADF4(C16) will enable the tailored design of functional spider silk‐based biomaterials in the future. It will be especially useful in context of chemical modifications and genetic fusions supporting the development of fibril‐based hydrogel systems in the field of biosensing and tissue engineering.