Fig. 1.
Refolding of NBD investigated by optical tweezers experiments. (A) Optical tweezers assay setup. EcNBD protein is tethered N and C terminally to dsDNA handles between two trapped beads. The mobile trap can be moved to exert a force on the molecule. (B) Series of measured stretching cycles: a fingerprint of the native protein (pink), and refolding intermediates (RFI1, red circle; RFI2, yellow star). Force-extension traces were recorded at a pulling speed of 200 nm/s and waiting time at zero force of 1 s. (C) The plot of natively refolded fraction dependent on the waiting time at zero force [EcNBD-nc (pink) and mtNBD (green)]. Fitting the data with a simple exponential equation gave a refolding rate for EcNBD-nc of 0.03 ± 0.02 s−1; mtNBD does not refold in our experiments. (D) Contour lengths measured for the refolding intermediates RFI1 (red circle) and RFI2 (yellow star). Native unfolding (gray) shows, first, stretching of DNA (LC = 0 nm), followed by the whole NBD unfolding (LC = 134 nm). (E) Scatter plot of all unfolding events (n = 10 molecules). Unfolding of mature NBD (pink) is characterized by an unfolding force of 34 pN and a contour length change of ∆LC = 134 nm (15). The refolding intermediates RFI1 (red circle) and RFI2 (yellow star) unfold at ∼5 and ∼7 pN, respectively. (F) Force-extension traces of the unfolding of isolated lobe II (aa 183–383). Unfolding of intact lobe II (pink, left), the next stretching shows the RFI1 and RFI2 unfolding (red circle and yellow star, right). (G) Scatter plot of the unfolding events in NBD 183–383. (H) Illustration of analyzed loop insertion variants Ins183 (K183-L20), Ins290 (A290-L20), and Ins364 (D364-L20). (I) Contour length transformation plots of EcNBD-nc, Ins290, Ins183 and Ins364 NBD variant. Ins183 and Ins364 show the same contour length for RFI1 and RFI2 as the EcNBD protein. The contour length differences for U-RFI1 in InsA290 are increased compared with EcNBD-nc (30.3 vs. 23.9 nm).