Study The Mechanical Properties Of A Single CsgA Protein With Nanometer Resolution

This paper contains two main parts: First,we have obtained a high-performance,multifunctional magnetic tweezers system by upgrading the software,hardware and algorithms of the magnetic tweezers;second,we use the upgraded magnetic tweezers to measure the monomeric CsgA protein molecules and obtain the force spectrum of CsgA molecules and ?helices structure.As one of the main tools of single-molecule mechanics research methods: magnetic tweezers,with its powerful flexibility and convenient maneuverability,has a wide range of applications in biomechanics.Improving the resolution of magnetic tweezers so that they can adapt to a wide range of testing situations has always been an important topic for singlemolecule research.First,we will replace the hardware of crucial units to enhance their ability to resist environmental noise and calculation speed;secondly,we will update the computer system to adapt to the new replacement parts;finally,the magnetic tweezers control software will be upgraded and optimization,including: cross-correlation algorithm upgrade,driver update,image optimization,quadrant interpolation algorithm embedding.After the upgrade is completed,the upper limit of the force measurement of the magnetic tweezers reaches near 200 p N,which basically covers all the force fields in the single-molecule biological process.The upgraded cross-correlation algorithm can enable the sampling frequency to reach 200 Hz,and its measurement accuracy can also be stabilized at the nanometer level,and the accuracy of the quadrant interpolation algorithm can even reach the sub-nanometer level.This constructed high-precision,multi-functional magnetic tweezers system will be used for the subsequent measurement of CsgA monomer.The CsgA protein derived from E.coli biofilm can self-assemble into nanofibers and is widely used in molecular materials.At present,there have been molecular dynamics for the mechanical study of a single CsgA molecule,and there is a lack of experimental measurement of a single CsgA molecule.We use a single CsgA molecule as the test object,and the upgraded magnetic tweezers can clearly capture the signal of a single ?-sheet unfolding.The work required to fully unfolding single CsgA and the length of the ?-helix unfolding of the structure are studied by changing force stretching and constant force stretching.The unfolding extension are distributed around 10 nm,13nm and 16 nm.The difference in these signals may be caused by the different positions of the ?-sheets.At the same time,many multi-unfolding signals were observed,and these multi-unfolding signals were also observed in CsgA self-assembled fibers.The work-elongation curve obtained in the experiment is similar to the Steered Molecular Dynamics(SMD)result.This study is helpful to the development of magnetic tweezers technology such as reducing the cost of magnetic tweezers,improving the accuracy of magnetic tweezers,and enriching the functions of magnetic tweezers.It can be used for the measurement of many single proteins and nano-level DNA.At the same time,the research deepened the understanding of the structure,function and application of biomaterials of CsgA.