Force Spectroscopy And Imaging Of Single CsgA Nanofiber

CsgA,a major protein subunit of E.coli biofilms,plays key roles in bacterial adhesion and invasion.In addition,it can also self-assemble into amyloid nanofiber.Csg A nanofiber possesses high stiffness,strength and mechanical rigidity due to its characteristic cross-? hydrogen-bonded structures,known as amyloid structures.Their important biological roles and promising applications in material science make it essential to investigate the mechanical properties of Csg A nanofibers.Although there have been some recent advances,few experimental studies have focused on the mechanical properties of single Csg A nanofibers.In addition,single molecule techniques such as atomic force microscopy,optical tweezers,and magnetic tweezers have been widely applied to study proteins.In these studies,the linking of proteins is usually achieved through a tag such as histidine,cysteine,or biotin.These proteins need to be chemically conjugated or genetically constructed,which is time consuming and lacks efficiency.Here we have established a simple method to measure all kinds of proteins without labels.The carboxyl terminus of a protein is attached to the amine group on a magnetic bead,and the amine terminus of the protein is attached to glutaraldehyde on the glass slide.The force spectrum and imaging of a single Csg A nanofiber were studied by magnetic tweezers experiment and scanning electron microscopy(SEM)using a tag-free linking method.Then the single Csg A nanofibers were screened by the relationship between the rotation number of magnet and the extension of the nanofibers.Next,scanning electron microscopy experiments were performed.It was directly observed that one end of a single nanofiber was connected to magnetic beads and the other end was connected to the glass.The diameter distribution of Csg A nanofibers was obtained by statistical analysis of the SEM images.From the force-extension curves of Csg A nanofibers,we demonstrate that the Csg A nanofibers follow the wormlike chain model and have the corresponding properties.In addition,the distribution of the quarter power of the persistence length and the distribution of the diameters are consistent.The bending stiffness of the nanofiber depends on the fourth power of its diameter.The number of filaments in nanofibers can be estimated based on the persistence length analysis.We also found that a single nanofiber was able to extend 17.1 times,indicating excellent tensile strength and rich potential for applications.Finally,we studied the extensions of single Csg A nanofibers under a constant magnetic force.Nanofibers which have relatively large changes of extensions are prone to show jumping steps.Our results provide fundamental information for the design and application of curli nanofibers in synthetic biology and materials science.