Magnetic tweezers were used to study the force spectra of CsgA, a major protein subunit of Escherichia coli biofilms, at fibril level.

The CsgA protein in Escherichia coli can self-assemble into an amyloid nanofiber, making it a promising biomaterial capable of resisting harsh environments. The assembling and unfolding mechanism of a single CsgA molecule is essential for all applications of CsgA materials but remains poorly understood. CsgA-CBD and Mfp5-CsgA are chimeric proteins generated by introducing the chitin-binding domain (CBD) and mussel foot protein 5 (Mfp5) into CsgA. In this study, we used magnetic tweezers to study the mechanical properties of a single CsgA molecule and the CsgA-CBD and Mfp5-CsgA assembled nanofibers. Our results demonstrated that the unfolding spectra of five β-sheets of a single CsgA molecule were not uniform and that several β-sheets simultaneously broke after an external force was applied. We applied ∼331 kcal/mol of work, via mechanochemical coupling, to unfold CsgA to a 94.6% strain. The average persistence lengths of the CsgA-CBD and Mfp5-CsgA nanofibers were 2.7 and 4.5 nm, respectively, while Young’s moduli of the two nanofibers were 42.8 and 56.1 MPa, respectively, both of which were less than those of the CsgA nanofiber. A relatively high force was required to unfold some of the nanofibers, which indicated that mechanical stimuli could help remove amyloid particles. This study assesses the mechanical properties of a single CsgA and lays the foundation for its subsequent use as a biological material and provides new insights into mechanochemical coupling nanofibers to design materials with tunable functions.