SKM 2023 – wissenschaftliches Programm
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BP: Fachverband Biologische Physik
BP 23: Single Molecule Biophysics
BP 23.4: Vortrag
Donnerstag, 30. März 2023, 10:30–10:45, BAR 0106
Complex unfolding, refolding and DNA association of the relaxase TrwC — •César Augusto Quintana-Cataño1, Miriam Schramm1, Ekaterina Vorobevskaia1, Andreas Hartmann1, and Michael Schlierf1,2 — 1B CUBE - Center for Molecular Bioengineering, TU Dresden, Germany — 2Cluster of Excellence Physics of Life, TU Dresden, Germany
Referred as "bacterial sex", bacterial conjugation is a process in which a donor cell transfers DNA to a recipient cell. Conjugation is a main driver for spreading of antibiotic resistance genes. The relaxase TrwC associated to the type 4 secretion system is a multi-domain model enzyme essential for bacterial conjugation. It serves multiple purposes: DNA recognition, nicking and unwinding in the donor cell; and re-ligation of ssDNA in the receptor cell. For this last step a mechanical unfoldase denaturates TrwC, which then refolds in the receptor cell. TrwC is a multi-domain protein consisting of 966 amino acids with a 55 amino acids long intrinsically disordered C-terminus. Here, we studied unfolding and folding mechanics of TrwC using single-molecule magnetic tweezers force spectroscopy. We show that the subdomains of TrwC unfold at distinct, different force regimes ranging from ~10 pN to 90 pN and TrwC refolds in a complex multistep reaction. Using our data, we can quantify the folding energetics of TrwC. We have further discovered complex DNA binding events of monomers and dimers using fluorescence correlation spectroscopy, allowing us to build a model of DNA association. We anticipate that our data helps to understand a key enzyme of DNA conjugation and its multifold activities.